CD8 binding agents and uses thereof

Abstract

To provide a pharmaceutical composition for use in a method for predicting the responsiveness of a subject having an autoimmune disease or condition, transplant rejection, or graft versus host disease to an immunotherapeutic agent.SOLUTION: The pharmaceutical composition comprises a labeled CD8-binding agent comprising a heavy chain variable domain (VHH domain) of a heavy chain antibody, wherein the method comprises a) administering the labeled CD8-binding agent to the subject; and b) detecting binding of the labeled CD8-binding agent to CD8 + T cells in a diseased tissue of the subject, wherein detection of the binding indicates that the subject is likely to respond to the immunotherapeutic agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 895,865, filed September 4, 2019. This application claims priority to US Provisional Patent Application No. 2005 / 0129994, the contents of which are incorporated herein by reference in their entirety. do.

[0002] ASCII text file sequence listing submission The contents of the following ASCII text file submissions are incorporated herein by reference in their entirety: Incorporated: Sequence Listing in Computer Readable Format (CRF) (Filename: 1463920 49240SEQLIST.txt, Recorded on August 19, 2020, Size: 14KB ).

[0003] The present application relates to CD8 binding agents based on anti-CD8 VHH domains, and in vivo CD8 in vo + To use such CD8 binding agents to image T cells This relates to the method. [Background technology]

[0004] Characterization of the number, type, and spatial distribution of immune cells in tumor tissues is crucial for cancer diagnosis. , which can provide important information regarding prognosis, therapy selection, and response to therapy. Specifically, CD8 + Cytotoxic lymphocytes play a diagnostic and prognostic role in various cancers. CD8 has been consistently reported to be of great importance. + Current methods for detecting cells The method involves isolating cells from peripheral blood or tissue of interest. is error-prone and CD8 + Cell number, localization, and migration are monitored in vivo. It does not provide dynamic information that reflects the immune cells in vivo. A representative non-invasive method is positron emission tomography (PET) using radiolabeled tracers. However, the use of such tracers is hindered by the half-life of the radioisotope and In vivo, cell division leads to probe dilution. There are known in the art CD8 + Monitor changes in cell abundance and temporal distribution in vivo There remains a need for methods and reagents for doing so. Summary of the Invention

[0005] As used herein, a CD8-binding antibody comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain) is agonists having a K of about 1 nM or lower D C specifically binds to human CD8 In some embodiments, the CD8 binding agent is about 500 pM or less, about 250 pM or less, or about 100 pM M or lower K D In some embodiments, The CD8 binding agent has a K of about 132 pm or about 50 pM. D specifically binds to human CD8 In some embodiments, the CD8 binding agent has a binding activity of about 0.002 / sec or less. k lower than or about 0.001 / sec or lower off Binds to human CD8 In some embodiments, the CD8 binding agent has a β-glucosidase activity of about 0.0018 / sec or about 0.0 0085 / sec k off In some embodiments, the CD8 binding molecule binds to human CD8. The agent has a K of about 1 nM or lower.D It binds to cynomolgus monkey CD8. In embodiments, the CD8 binding agent has a cytotoxicity of about 500 pM or less, about 250 pM or less. M or lower, or K of about 150 pM or lower D crabeater In some embodiments, the CD8 binding agent binds to monkey CD8. is approximately 137 pM D In some embodiments, the CD8 binding The combined agent may have a rate of about 0.004 / sec or less, or about 0.002 / sec or less. Or lower k off In some embodiments, C D8 binding agents have a k of about 0.0037 / sec or about 0.0019 / sec off Dekaniku In some embodiments, the CD8 binding agent binds to monkey CD8. a CD8 binding half-life (e.g., 1 hour, 2 hours, or longer) of at least about 30 minutes In some embodiments, the antibody has a CD8 binding domain. The combined agent has a K of about 1 nM or less. D Specific binding to rhesus monkey CD8 In some embodiments, the CD8 binding agent binds to both mouse CD8 and rat CD8. Doesn't fit.

[0006] In some embodiments according to (or as applied to) any of the above embodiments, the CD8 binding The synergistic agent is CD8 + It neither stimulates nor inhibits T cell activation. The D8 binding agent binds to CD8 + In some embodiments, the CD8+ receptor does not induce T cell proliferation. The synergistic agent is CD4 + Does not bind to T cells.

[0007] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main VHH domain is a camelid VHH, such as a llama VHH. In some embodiments, the VHH is chimeric. In some embodiments, the VHH is humanized. The VHHs are affinity matured.

[0008] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main ones are Arg25, Lys42, Gln44, Val45, Leu46, and Leu47. , Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu Specific for human CD8α epitopes including 94, Gly95, Asp96, and Thr97 In some embodiments, the human The amino acid residues of the CD8α epitope are those of a CD8-binding agent or is approximately one or more amino acid residues of the VHH domain in the crystal structure of the VHH domain. It is within 4.5 Å.

[0009] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main complementarity determining region (CDR) 1 comprises the amino acid sequence of SEQ ID NO: 6 or 7; CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and any one of SEQ ID NOs: 10 to 12 The CDR3 comprises one amino acid sequence.

[0010] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main CDR1 comprises the amino acid sequence of SEQ ID NO: 6, and the main CDR2 comprises the amino acid sequence of SEQ ID NO: 8. and CDR2 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments according to (or as applied to) either, the VHH domain is CDR1 comprising the amino acid sequence of SEQ ID NO:6, CDR2 comprising the amino acid sequence of SEQ ID NO:9, and and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VHH domain has the amino acid sequence of SEQ ID NO: 7. CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 11 A CDR3 comprising the amino acid sequence of In some embodiments, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, the sequence CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR comprising the amino acid sequence of SEQ ID NO: 12 Includes 3.

[0011] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main comprises L49A, where the numbering is according to Kabat numbering. The CD8 binding agent is purified using protein A affinity chromatography. It's fine.

[0012] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main ones are V89T substitution, T110Q substitution, S112Q substitution, and A addition at position 114. (hereinafter referred to as "A114 addition") In some embodiments, the VH may include amino acid modifications, in which case the numbering is according to Kabat numbering. The H domain contains V89T, T110Q, and S112Q substitutions, and an A114 addition. In some embodiments, the numbering is according to Kabat numbering. The agent does not bind to pre-existing anti-VHH antibodies in the subject receiving the CD8 binding agent.

[0013] In some embodiments according to (or adapted from) any of the above embodiments, the VHH driver The main comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain The main comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments where applicable, the VHH domain comprises the amino acid sequence of SEQ ID NO: 4. .

[0014] Also herein, CD8 binding according to (or applied to) any of the above embodiments is In some embodiments, an isolated nucleic acid encoding the combined agent is provided. Expression vectors containing nucleic acids according to (or adapted from) any of the forms are provided. In an embodiment of the present invention, a nucleic acid or expression vector according to (or adapted from) any of the above embodiments is A host cell comprising the vector is provided. In some embodiments, the host cell is a mammalian cell. , e.g., eukaryotic cells such as CHO cells or Expi293 cells. In the present case, the host cell is a prokaryotic cell, such as an E. coli cell.

[0015] As used herein, a CD8 binding antibody according to (or adapted from) any of the above embodiments is A method for making a pharmaceutical preparation, comprising: a) applying a composition according to any of the above embodiments (or a) culturing the host cells under conditions in which the agent is produced; and b) culturing the host cells Further provided are methods comprising recovering the CD8 binding agent produced by the cells. do.

[0016] In some embodiments according to (or as applied to) any of the above embodiments, the CD8 binding A CD8 binding agent that includes a label is referred to herein as a "labeled CD8 binding agent." These are called "8-binding agents."

[0017] In some embodiments, the method for preparing a labeled CD8 binding agent comprises: The hydroxyl group moiety may be a hydroxyl group moiety of a CD8 binding agent according to (or adapted from) any of the above embodiments. conjugating the VHH domain to provide a conjugate; Jugate, 18 The labeled CD8 binding antibody was contacted with an aluminum fluoride complex containing F. providing an agent, the agent comprising a chelating moiety having the formula (I):

[0018] [ka] In some embodiments, the conjugate is a compound of formula (I). one or more antioxidant compounds, such as tryptophan and / or N-acetyl-tryptophan The aluminum fluoride complex is contacted with the aluminum fluoride complex in the presence of

[0019] As used herein, a compound according to any of the above embodiments (or A labeled CD8 binding agent comprising an anti-CD8 VHH domain is provided. In some embodiments, the label is a fluorescent dye, a radionuclide, or an enzyme. In some embodiments according to (or applied to) any of In some embodiments, the radionuclide is 18 F, 89 Zr, 99m Tc, 67 Ga, 68 Ga, 64 Cu, 52 Mn, 111 In, or 124 I. In some embodiments, The VHH domain is conjugated to a label via a chelating moiety. In this form, the chelating moiety is covalently linked to the VHH domain via a lysine residue. In some embodiments, the label forms a complex with a metal, and the complex is bound by a chelating moiety. In some embodiments, the label is 18 F and the metal is aluminum In some embodiments, the chelating moiety is a compound of formula (I).

[0020] As used herein, CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 9 and CDR3 comprising the amino acid sequence of SEQ ID NO: 11. A labeled CD8 binding agent comprising a VHH domain, wherein the VHH domain is linked to a chelating moiety. via radionuclides (e.g. 18 F) conjugated to a labeled CD8-binding In some embodiments, the chelating moiety is a compound of formula (I): Radionuclides are complexed with aluminum 18 In some embodiments, the VHH domain is F. The main portion contains the amino acid sequence of SEQ ID NO:3.

[0021] As used herein, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 9 and CDR3 comprising the amino acid sequence of SEQ ID NO: 12. A labeled CD8 binding agent comprising a VHH domain, wherein the VHH domain is linked to a chelating moiety. via radionuclides (e.g. 18 F) conjugated to a labeled CD8-binding In some embodiments, the chelating moiety is a compound of formula (I): Radionuclides are complexed with aluminum 18 In some embodiments, the VHH domain is F. The main portion contains the amino acid sequence of SEQ ID NO:4.

[0022] Also herein, CD8 binding according to (or applied to) any of the above embodiments is A pharmaceutical comprising a combined agent (including a labeled CD8 binding agent) and a pharmaceutically acceptable carrier. A composition is provided.

[0023] The present specification provides a method for treating or diagnosing a disease or condition of interest using the above embodiments. CD8 binding agents (including labeled CD8 binding agents) and in the preparation of a medicament for treating or diagnosing a disease or condition of interest. a CD8 binding agent (labeled C) according to (or adapted from) any of the above embodiments, Further provided are uses of a D8 binding agent.

[0024] As used herein, a CD8 binding antibody according to (or adapted from) any of the above embodiments is a pharmaceutical comprising an active ingredient (including a labeled CD8 binding agent) and one or more antioxidant compounds Formulations are further provided. In some embodiments, the one or more antioxidant compounds are methyl In some embodiments, the pharmaceutical In some embodiments, the pharmaceutical agent comprises methionine and N-acetyltryptophan. The formulation further comprises histidine and sucrose.

[0025] As used herein, the subject's CD8 + A method for detecting cells, comprising: a) the above embodiment; A step of administering a labeled CD8 binding agent to a subject, the step being performed by (or as applied to) one of the following: and b) binding the labeled CD8 binding agent to the subject's CD8 + A stabilizing agent that detects binding to cells The binding detection was performed using CD8 + A method is provided for indicating the presence of a cell. In this state, the labeled CD8 binding agent and the subject's CD8 + The step of detecting binding to the cell includes: Subject's CD8 + In some embodiments, the subject's CD8 + cell The imaging is performed by subjecting the subject to a positron emission tomography (PET) scan or positron emission tomography (PET) scan. This includes performing a PET / CT scan. In this embodiment, CD8 + The cells are CD8 + In some embodiments, the CD8 + Thin In some embodiments, the detecting step occurs about 1 hour after administration. Within a day or less (e.g., approximately 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour) In some embodiments, the method is performed within 10 minutes, 30 minutes, or less. The method is repeated one or more times, such as about 1-4 times per year. is repeated about 1 day after the previous administration of the CD8 binding agent. In some embodiments, the method is repeated for a period of time greater than one year. In some embodiments, the subject is a human or non-human spirit. In some embodiments, the subject is a cynomolgus monkey or a rhesus monkey. In some embodiments, the subject is a human. In some embodiments, the subject has cancer. In embodiments, the subject has an autoimmune disease or condition, transplant rejection, or graft-versus-host disease. Has.

[0026] As used herein, the present invention relates to a method for treating a cancer patient's response to an immunotherapeutic agent, cell therapy, or cancer vaccine. 10. A method for predicting responsiveness to a subject, comprising: a) administering to said subject a method for predicting responsiveness to a subject in accordance with any of the above embodiments (and (where applicable) administering a labeled CD8 binding agent to a subject; and (b) administering a labeled CD8 binding agent to a subject. 8-binding agents and CD8 in tumor tissue of interest + detecting binding to T cells , detecting binding can indicate whether a subject is likely to respond to an immunotherapeutic agent, cell therapy, or cancer vaccine. In some embodiments, methods are provided in which the antibody is coupled to a labeled CD8 binding agent and shows that the antibody is highly Elephant CD8 + The step of detecting binding to the cells may be performed using a CD8 + Imaging cells In some embodiments, the subject's CD8 + Cell imaging involves positron emission PET scan or positron emission tomography / computed tomography (PET) In some embodiments, the method includes (c) performing a CT scan. A therapeutically effective amount of an immunotherapy agent, cell therapy, or cancer vaccine is administered to the identified subject. In some embodiments, the detecting step is performed within about 1 day or less after administration. or less (e.g., about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes) In some embodiments, the method is performed within a year. In some embodiments, the method is repeated one or more times, such as about 1 to 4 times. In some embodiments, the administration of the binding agent is repeated at least one day after the previous administration. The method is repeated over a period of more than one year.

[0027] Also provided herein are methods for monitoring disease progression in a subject with cancer, a) a labeled CD8 binding agent according to (or adapted from) any of the above embodiments and b) administering to a subject a labeled CD8+ antibody at a first time point and a second time point. Combined agent and CD8 of target tumor tissue + detecting binding to a T cell In some embodiments, a labeled CD8 binding agent and a subject's CD8 + Cell binding The step of detecting the presence of CD8 + In some embodiments, the method further comprises imaging the cells. So, the target CD8 + Cell imaging involves subjecting a subject to positron emission tomography (PET) scanning. A PET / CT scan or positron emission tomography / computed tomography (PET / CT) scan will be performed. In some embodiments, the method includes (c) administering a therapeutically effective amount of an immunotherapeutic agent, a cellular therapy agent, or a combination thereof. The method further comprises administering a cancer vaccine to a subject, and measuring the tumor at the second time point. CD8 in tumor tissue + T cell levels were measured using CD8 T cells in tumor tissue at the first time point. + T cell In some embodiments, the detecting step further comprises a step of detecting a signal higher than the threshold. Within about 1 day or less after administration (e.g., about 6 hours, 4 hours, 2 hours, 90 In some embodiments, the method is performed within 1 minute, 1 hour, 30 minutes, or less. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. Alternatively, the method is repeated at least one day after the previous administration of the CD8 binding agent. In some embodiments, subjects are monitored for longer than one year.

[0028] As used herein, a patient who has received or is currently receiving an immunotherapeutic agent, cell therapy, or cancer vaccine is considered to be a patient who has not received an immunotherapeutic agent, cell therapy, or cancer vaccine. 1. A method for monitoring the treatment progress of a subject with cancer, comprising administering to a subject: i) an immunotherapeutic agent; In conjunction with cell therapy or cancer vaccines, according to any of the above embodiments (or applications) ii) administering to the subject a labeled CD8 binding agent at a first time point; and and at a second time point, the labeled CD8 binding agent and the CD8 binding agent in the tumor tissue were + Binding to T cells In some embodiments, a method is provided that includes detecting a labeled CD8 binding agent. Agent and subject's CD8 + The step of detecting binding to the cells may be performed using a CD8 + Imaging cells In some embodiments, the subject's CD8 + Cell imaging involves the use of positively charged particles Electron Emission Tomography (PET) scan or Positron Emission Tomography / Computed Tomography ( In some embodiments, the method further comprises performing a PET / CT scan. The agent is administered prior to the immunotherapy, cell therapy, or cancer vaccine, and the first time point is Administration of the labeled CD8-binding agent followed by administration of an immunotherapy, cell therapy, or cancer vaccine The first time point is before administration of the immunotherapy agent, cell therapy, or cancer vaccine, and the second time point is after administration of the immunotherapy agent, cell therapy, or cancer vaccine. In some embodiments, the immunotherapeutic, cell therapy, or cancer vaccine comprises a labeled CD8-binding The first time point is administered before the administration of the immunotherapeutic agent, cell therapy, or cancer vaccine. and after administration of a labeled CD8 binding agent, the second time point being a time interval between the first time point and the second time point. In some embodiments, the detecting step occurs within about 1 day or less after administration. Within a short period of time (for example, within approximately 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or In some embodiments, the method is performed about 1-4 times per year. In some embodiments, the method comprises: In some embodiments, the subject is administered one or more doses of the agent at least one day after the previous administration of the agent. monitored for a period longer than one year.

[0029] by any of the above predictive or monitoring methods (or as appropriate) In some embodiments, an immunotherapeutic agent is administered to the subject. Immunotherapeutic agents include anti-PDL1 antibodies, anti-PD1 antibodies, anti-TIGIT antibodies, and TIGIT antagonisms. anti-CSF-1R antibody, anti-CSF-1R antagonist, anti-CEA antibody, anti-CEA antagonist, anti-CTLA4 antibody, CTLA4 antagonist, anti-OX40 antibody, or In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is in combination with one or more therapeutic agents. In some embodiments, the one or more therapeutic agents are administered in a manner consistent with TARCEVA (registered trademark). ® (erlotinib), ZELBORAF® (vemurafenib), GAZY VA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (Benib) rafenib) and COTELLIC® (cobimetinib), ALECENSA (Alectinib), KADCYLA (Ado-trastuzumab) HERCEPTIN® (trastuzumab), PERJETA® (registration) (Registered trademark) (pertuzumab), polatuzumab, IFN-alpha, anti-CD40, anti-OX4 0 antibody, OX40 agonist, anti-CSF-1R antibody, anti-CEA antibody, IDO inhibitor, and In some embodiments, the immunotherapeutic agent is an anti-TIGIT antibody. In some embodiments, the cytokine is IL2, genetically engineered IL2, IL15, or genetically engineered IL15. In some embodiments, the immunotherapeutic agent specifically binds to CD3. In some embodiments, the bispecific antigen-binding molecule comprises: In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antigen-binding molecules The original binding molecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the immunotherapeutic agent is an antigen-binding molecule that specifically binds to CD16A. is a dendritic cell modulator such as a dendritic cell activator or dendritic cell growth factor.

[0030] by any of the above predictive or monitoring methods (or as appropriate) In some embodiments, a cancer vaccine is administered to the subject. The cancer vaccine is a personalized cancer vaccine (PCV).

[0031] by any of the above predictive or monitoring methods (or as appropriate) In some embodiments, a cell therapy is administered to the subject. In some embodiments, the cell therapy is a CAR-T. In some embodiments, the cell therapy is a neoantigen-specific T cell is.

[0032] As used herein, a subject having an autoimmune disease or condition, transplant rejection, or graft-versus-host disease A method for predicting a subject's responsiveness to an immunotherapeutic agent, comprising: a) administering to a subject a therapeutic agent according to any of the above embodiments; administering to a subject a labeled CD8 binding agent according to (or as applied to) either and b) binding a labeled CD8 binding agent to CD8 in diseased tissue of a subject. + Binding to T cells detecting binding, wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. In some embodiments, a method is provided in which a labeled CD8 binding agent and a subject's CD8 binding agent are combined. 8 + The step of detecting binding to the cells may be performed using a CD8 + This includes imaging the cells. In some embodiments, the subject's CD8 + Cell imaging is performed using positron emission tomography (PET). (PET) scan or positron emission tomography / computed tomography (PET / CT) In some embodiments, the method further comprises (c) performing a scan where binding is detected. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immunotherapeutic agent. The detecting step may be performed within about 1 day or less (e.g., about 6 hours) after administration. within 1 hour, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method includes administering at least one CD8 binding agent to a subject after a previous administration of the CD8 binding agent. In some embodiments, the method is repeated for longer than a year. It will be returned.

[0033] Also used herein are autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease. A method for monitoring disease progression in a subject having a disease comprising: a) administering to a subject a therapeutic agent selected from the group consisting of a steroid hormone, ... administering to the subject a labeled CD8 binding agent according to (or adapted for) and b) combining the labeled CD8 binding agent with the diseased tissue of the subject at a first time point and a second time point. CD8 + detecting binding to T cells from the first time point and the second time point; CD8 + Increased T cells may be a contributing factor to autoimmune diseases or conditions, transplant rejection, or graft-versus-host syndrome. In some embodiments, a method is provided for detecting a CD8-binding protein that is a target of the antibody, the antibody being in a state where the antibody is a CD8-binding protein, and the antibody being in a state where the antibody is a CD8-binding protein. Drugs and subject's CD8 + The step of detecting binding to the cells may be performed using a CD8 + Imaging cells In some embodiments, the subject's CD8 + Imaging of cells is a positive Electron emission tomography (PET) scan or positron emission tomography / computed tomography In some embodiments, the method further comprises: (c) performing a PET / CT scan; The method further comprises administering to the subject a therapeutically effective amount of an immunotherapeutic agent, wherein the method further comprises administering to the subject a therapeutically effective amount of an immunotherapeutic agent, the method comprising administering to the subject a therapeutically effective amount of an immunotherapeutic agent, the method comprising administering to the subject a therapeutically effective amount of an immunotherapeutic agent, the method further ... CD8 in affected tissue + T cell levels were measured using CD8 + T cell In some embodiments, the detecting step further comprises detecting a level lower than the threshold. Within about 1 day or less after administration (e.g., about 6 hours, 4 hours, 2 hours, 90 In some embodiments, the method is performed within 1 minute, 1 hour, 30 minutes, or less. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. Alternatively, the method is repeated at least one day after the previous administration of the CD8 binding agent. In some embodiments, subjects are monitored for longer than one year.

[0034] As used herein, an autoimmune disease or condition that has received or is receiving an immunotherapeutic agent, A method for monitoring the treatment progress of a subject with transplant rejection or graft-versus-host disease. i) in conjunction with an immunotherapeutic agent according to (or adapted from) any of the above embodiments ii) administering a labeled CD8 binding agent to a subject; and At time point 2, the labeled CD8 binding agent and CD8 in the diseased tissue were + Detecting binding to T cells In some embodiments, a method is provided comprising the steps of: CD8 + The step of detecting binding to the cells may be performed using a CD8 + Imaging cells In some embodiments, the subject's CD8 + Cell imaging involves the use of positron emission cross-sections of a subject. Positron Emission Tomography (PET) scan or Positron Emission Tomography / Computed Tomography (PET / In some embodiments, the labeled CD8 binding agent is , administered before the immunotherapeutic agent, and the first time point is after administration of the labeled CD8 binding agent but after the immunotherapy. The first time point is before administration of the therapeutic agent, and the second time point is after administration of the immunotherapeutic agent. The immunotherapeutic agent is administered before the labeled CD8 binding agent, and the first time point is the time and the second time point is after administration of the labeled CD8 binding agent, ... In some embodiments, the detecting step occurs within about 1 day or less after administration. Within a shorter period (for example, within approximately 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or In some embodiments, the method is performed within about 1-4 years. In some embodiments, the method is repeated one or more times, such as once. In some embodiments, the subject is administered a dose of the active ingredient in a medicament for at least one day after the previous administration of the active ingredient. Monitored for longer than one year.

[0035] Provided herein are gut microbial strains associated with responsiveness to treatment with immunotherapeutic agents. 1. A method for identifying a gut microbiome from a population of subjects with cancer, comprising: a) identifying a gut microbiome from a population of subjects with cancer; obtaining a population sample, the population comprising subjects who are responsive to treatment with an immunotherapeutic agent and and b) subjects who are responsive to treatment with an immunotherapeutic agent; Gut microbiome samples from subjects and those not responsive to treatment and c) analyzing intestinal samples associated with subjects who are responsive to the treatment. and identifying the microbial strain, and the responsiveness is according to any of the above embodiments. or applied) labeled CD8 binding agent and CD8 in the tumor tissue of the subject. + Binding to T cells and detecting binding indicates that the subject is responsive to the immunotherapeutic agent. In some embodiments, the method comprises determining whether a patient is responsive to an immunotherapeutic agent. and preparing a microbiome-based drug containing a gut microbial strain associated with In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody. In embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody, such as atezolizumab.

[0036] As used herein, a labeled CD8 binding agent, etc., according to any of the above embodiments (also Kits and articles of manufacture containing the CD8 binding agents are further provided. In this embodiment, the kit or article of manufacture comprises a CD8 binding antibody according to any of the above methods. The instructions for using the preparation are included. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 provides an alignment of the amino acid sequences of exemplary anti-CD8 VHH domains, including llama VHH wt2C8 (SEQ ID NO: 1), humanized VHHs hu2C8v130 (SEQ ID NO: 2), hu2C8v142 (SEQ ID NO: 3), and hu2C8v144 (SEQ ID NO: 4), and non-binding control 2C8v145 (SEQ ID NO: 5). [Figure 2] FIG. 1 provides an alignment of the amino acid sequences of human CD8a (SEQ ID NO: 13), cynomolgus CD8a (SEQ ID NO: 14), and rhesus CD8a (SEQ ID NO: 15). [Figure 3] FIG. 1 shows the results of experiments performed to evaluate CD8+ cell specific binding of VHH-Fc variants in comparison to OKT8-Fc. [Figure 4] Figure 1 shows exemplary results of staining a whole blood cell sample from a healthy volunteer with 2C8 VHH. OKT8 is an anti-CD8 IgG and serves as a positive control. 3E8 VHH is a non-binding negative control. [Figure 5]Schematic of the crystal structure of 2C8 VHH. The structure on the left shows 2C8 VHH (light gray) bound to a CD8α / 8α homodimer (black, epitope highlighted in white; dimer reconstructed by crystallographic symmetry operations). The structure on the right shows the superposition of the 2C8 VHH:CD8α / 8α complex (same colors as in the left panel) with the published structure of an MHC class I complex with a CD8α / β heterodimer (PDB ID: 3DMM; MHC I is shown in light gray and CD8β is shown in medium-dark gray). [Figure 6] FIG. 1 shows the results of experiments performed to evaluate the binding of wild-type 2C8 and 2C8.v144 VHHs to pre-existing anti-VHH antibodies in blood samples of 96 healthy donors. [Figure 7A] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle). [Figure 7B] FIG. 1 provides the results of experiments performed to evaluate the CD8+ T cell protease release response to polyclonal T cell stimulation with anti-CD3 and anti-CD28 in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle). [Figure 7C] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation. [Figure 7D] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after stimulation with CEF peptide pools. [Figure 7E] FIG. 10 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after LPS stimulation. [Figure 8A]

[0023] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle), in which 10% FBS was used as the culture medium. [Figure 8B]

[0023] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle), in which 10% autologous donor plasma was used as the culture medium. [Figure 8C] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation, in which 10% FBS was used as the culture medium. [Figure 8D] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation, in which 10% autologous donor plasma was used as the culture medium. [Figure 9] FIG. 1 shows the results of experiments performed to evaluate the CD8 imaging capacity of 18F-anti-CD8 VHH in chimeric HPBALL / Daudi tumor xenograft mice. [Figure 10] Figure 1 shows PET MIPs of TALL1 tumor xenografted mice on day 5 (i.e., day 6) after injection (day 0) of 89Zr-OA mAb control or 89Zr-huOKT8.v1-OA (left), or 90 minutes after injection of 18F-control VHH or 18F-anti-CD8 VHH (right). [Figure 11] FIG. 1 shows PET MIP images of rhesus monkeys 1 hour after injection with 18F-anti-CD8 VHH (top row) or 18F-control VHH (bottom row). DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, a CD8 binding agent comprising a VHH domain (such as an anti-CD8 antibody or (including antigen-binding fragments of the antibody) that specifically bind to human CD8 with high affinity, but 8 + It neither stimulates nor inhibits T cells, and CD8 + CD8-binding agents that do not induce T cell proliferation The CD8 binding agents are provided in non-human primates, such as rhesus monkeys and cynomolgus monkeys. It is possible to bind with high affinity to CD8 of various types. Compared to the combined agents, the CD8 binding agents described herein have higher permeability and Therefore, the CD8 binding agents described herein have a shorter serum half-life than Within a short time frame after the drug (e.g., within 1 hour, 1 day) + Cells (e.g., C D8 + Suitable for detecting the presence, localization, and / or quantity of T cells) with same-day readout Repeated imaging and multiplex imaging in combination with other biomarkers In addition, the CD8 binding agents described herein can bind to CD8. High sensitivity, linear correlation with CD8 levels over a wide dynamic range, and permeability High accuracy due to reduced sensitivity to external factors and high performance in mouse xenograft models The images show high image quality reflected by a low tumor-to-blood ratio.

[0039] As used herein, in vivo CD8 + In a method for detecting T cells, Methods for using the 8-binding agents are provided. Also provided are methods for treating diseases (e.g., cancer, autoimmune diseases) immunotherapeutic agents for subjects with a comorbid disease or condition, transplant rejection, or graft-versus-host disease In a method for predicting responsiveness to treatment with a CD8 binding agent of the present invention, In addition, methods for using the CD8 binding agents herein are provided. , being treated with immunotherapeutic agents, or a disease (e.g., cancer, autoimmune disease or condition) and monitoring disease progression and / or treatment progress in subjects with graft-versus-host disease (e.g., graft rejection, or graft-versus-host disease). A method for monitoring is provided.

[0040] definition As used herein, the term "human CD8" refers to human cluster of differentiation 8 molecules. Proteins, polypeptides, or proteins corresponding to the 8th molecule of differentiation in humans refers to these parts. Full-length human CD8 acts as a co-receptor for the T cell receptor. The human CD8 protein is a dimer consisting of the CD8α chain and The term "human CD8" refers to a pair of CD8 chains, including CD8α / C and CD8β chains. CD8α homodimer, CD8α / CD8β heterodimer, CD8α chain, CD8β chain, or The terms "CD8a" and "CD8α" are used herein to refer to CD8α and CD8α-specific proteins, including portions thereof, such as the extracellular domain. Used interchangeably herein, "CD8b" and "CD8β" are used interchangeably herein. An exemplary sequence of the human CD8 α-chain is shown in FIG.

[0041] The term "CD8 binding agent" as used herein refers to any CD8 binding molecule. CD8 binding agents can be polypeptides, proteins, antibodies (four-chain antibodies or heavy-chain antibodies), antibody fragments (e.g., VHH), or human CD8, cynomolgus monkey CD8, and Immunoconjugates that bind to CD8 and / or other non-human CD8 proteins or peptides The CD8 binding agent may also be a small molecule label, such as a radionuclide. A CD8 binding agent that includes a label is referred to herein as a "labeled CD8." They are also called "8-binding agents."

[0042] The term "antibody" is used herein in the broadest sense and refers to any antibody having the desired antigen-binding activity. sex, i.e., CD8 (human CD8, cynomolgus CD8, and / or rhesus CD8 As long as the antibody exhibits binding to the antibody, it may include, but is not limited to, a monoclonal antibody, a monovalent antibody, etc. (e.g., one-armed antibodies, four-chain antibodies (e.g., IgG antibodies), heavy-chain antibodies, The term "four-chain antibody" encompasses various antibody structures, including antibodies and antibody fragments thereof. As used herein, refers to an antibody or antigen-binding fragment having two heavy chains and two light chains. are used synonymously to mean

[0043] An "antibody fragment" comprises a portion of an antibody, preferably the antigen-binding or variable region of the antibody. Examples of antibody fragments include VHH, single domain antibodies, Fab, Fab', F(ab')2, and Fv fragments: diabodies; linear antibodies (U.S. Pat. No. 5,641,870, Example 2; see Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. The term refers to the variable domain, as compared to the other part of the immunoglobulin that contains the antigen-binding site. The constant domain refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence. , C for heavy chain H 1. C H 2, and C H 3 domains (collectively referred to as C H ) and light chain CHL (or CL ) domain.

[0044] The term "Fc region" or "fragment crystallizable region" as used herein refers to a region of the Fc domain that is a fragment of a naturally occurring sequence. To define the C-terminal region of an immunoglobulin heavy chain, including the Fc region and variant Fc regions Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, human IgG heavy chains are The chain Fc region typically begins at amino acid residue Cys226, or begins at amino acid residue Pro230, or The Fc region is defined as extending to the carboxyl terminus of the C-terminal lysine (EU numbering system) of the Fc region. Residue 447 (by ATP) may be present, for example, during antibody production or purification, or when encoding the heavy chain of an antibody. The nucleic acid encoding the gene may be removed by recombinant genetic engineering. Intact antibody compositions include antibody populations with all K447 residues removed, antibody populations with K447 residues removed, and antibody populations with K447 residues removed. The non-depleted antibody population and a mixture of antibodies with and without the K447 residue Natural antibodies suitable for use in the antibodies described herein may include antibody populations having a mixture of natural and synthetic antibodies. The Fc region sequences include human IgG1, IgG2 (IgG2A, IgG2B), and IgG3 , and IgG4.

[0045] The term "monoclonal antibody" as used herein refers to a substantially homogeneous antibody. It refers to antibodies obtained from a population of antibodies in the body, i.e., the individual antibodies that make up the population are the most likely natural mutations present in the Monoclonal antibodies are highly specific and have a single directed to different antigenic sites. Typically directed to different determinants (epitopes). In contrast to polyclonal antibody preparations, which contain different antibodies attached to each monoclonal antibody, Antibodies are directed against a single determinant on an antigen. In addition to being specific, Monoclonal antibodies are produced by hybridoma cultures that are not contaminated by other immunoglobulins. The modifier "monoclonal" is advantageous in that it is synthesized by It characterizes antibodies as being obtained from a homogeneous antibody population and is suitable for production of antibodies by any particular method. For example, nothing in this application should be construed as requiring the use of Clonal antibodies can be produced by a variety of techniques, including, for example: Hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laborat ory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et a l., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 198 1), recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567), Page display techniques (e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Sidhu et al, J. Mol. Biol. 338(2): 2 99-310 (2004);Lee et al, J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132 (2004)), as well as human immunoglobulin loci In animals that have some or all of the genes encoding human immunoglobulin sequences techniques for producing human or human-like antibodies (e.g., WO 1998 / 2489 Pamphlet No. 3; Pamphlet No. WO 1996 / 34096; Pamphlet No. WO 199 6 / 33735; WO 1991 / 10741; Jako bovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993);Jakobovits et al, Nat ure 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US Pat. Specification No. 5,545,807; Specification No. 5,545,806; No. 5,569,82 Nos. 5,625,126, 5,633,425, and No. 5,661,016; Marks et al, Bio / Technology 10: 779-783 (1992); Lonb erg et al, Nature 368: 856-859 (1994);Morrison, Nature 368: 812-813 (1994);Fis hwild et al, Nature Biotechnol. 14: 845-851 (1996);Neuberger, Nature Biotechnol . 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1 995).

[0046] The term "variable region" or "variable domain" refers to the amino acid sequence of an antibody that is involved in binding to an antigen. Refers to the heavy or light chain variable domains of a natural antibody. The VH and VL domains generally have similar structures, with each domain containing four conserved frames. It contains a framework region (FR) and three hypervariable regions (HVRs) (see, e.g., Kindt et al. al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to specific antigens can be synthesized by combining VH or VL domains derived from antibodies that bind to the antigen. and isolate complementary VL or VH domain libraries, respectively. For example, Portolano et al., J. Immunol. 150:880-88 7 (1993); Clarkson et al, Nature 352:624-628 (1991).

[0047] The term "heavy chain antibody" also known as "heavy chain only antibody" or "HCAb" describes a functional antibody that contains two heavy chains but lacks the two light chains normally found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs. is known.

[0048] The term "single domain antibody" or "sdAb" refers to an antibody that contains three complementarity-determining regions (CDs). sdAb refers to a single antigen-binding domain containing the corresponding CDR alone. It is possible for the antibody to bind to an antigen without pairing with a polypeptide. Single domain antibodies are engineered from camelid HCAbs and designated "VHH" (defined below). Camelid sdAbs are among the smallest known antigen-binding antibody fragments (e.g., For example, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-2 6 (2013).

[0049] The term "VHH" or "variable domain of the heavy chain of a heavy chain antibody" refers to a single variable domain of a heavy chain antibody. VHH molecules refer to heavy chain variable domains of Camelidae species, e.g., camels, Derived from antibodies produced in llamas, vicuñas, dromedaries, alpacas, and guanacos A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR 2-CDR2-FR3-CDR3-FR4, and FR1 to FR4 are frames. CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0050] The term "hypervariable region" or "HVR" as used herein refers to a region in which a sequence is hypervariable. variable ("complementarity determining regions" or "CDRs") and / or structurally defined ("hypervariable loops"), and / or antigen contact residues ("antigen contacts"). "). Generally, four-chain antibodies and their The antigen-binding antibody fragment has six HVRs: three in the VH (H1, H2, and H3) and three in the VL. (L1, L2, L3). Generally, heavy chain antibodies contain three HVRs (HVR1, HVR2 , HVR3).

[0051] Several HVR definitions are in use and are encompassed herein. Exemplary HVRs of four-chain antibodies and their antigen-binding antibody fragments include the following: (a) amino acid residues 26-32 (L1), 50-52 (L2), 91-96 ( L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (b) hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) amino Noic acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50–65 (H2), and 95–102 (H3) CDRs (Kabat et al. al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Ser. Vice, National Institutes of Health, Bethesda, MD (1991); (c) amino acid residue 2 7c~36(L1), 46~55(L2), 89~96(L3), 30~35b(H1) , 47–58 (H2), and 93–101 (H3) (MacCallum et al. l. J. Mol. Biol. 262: 732-745 (1996)); and (d) HVR amino acid residues 46-5 6(L2), 47~56(L2), 48~56(L2), 49~56(L2), 26~3 5(H1), 26~35b(H1), 49~65(H2), 93~102(H3), and and combinations of (a), (b), and / or (c), including 94-102(H3).

[0052] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., PR Residues) are numbered herein according to Kabat et al., supra.

[0053] The amino acid residues of single domain antibodies (such as VHH) are determined by the method described in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195, the paper As applied to VHH domains, Kabat et al. ("Sequence of pr oteins of immunological interest", US Public Health Services, NIH Bethesda, Md., V granted by Publication No. 91 H Numbering can be done according to the domain's base numbering. According to this numbering, FR1 of VHH contains amino acid residues 1 to 30, and VHH CDR1 of VHH contains amino acid residues 31 to 35, and FR2 of VHH contains amino acid residues 36 to 49. CDR2 of VHH contains amino acid residues 50 to 65, and FR3 of VHH contains The CDR3 of VHH contains amino acid residues 66 to 94, and the CDR3 of VHH contains amino acid residues 95 to 102. and FR4 of VHH comprises amino acid residues 103 to 113. H As is well known in the art for VHH domains and VHH domains, each of the CDRs The total number of amino acid residues in the may not correspond to the total number of groups (i.e., one or more positions in the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may be It should be noted that the nucleotide sequence may contain more than the number of amino acid residues permitted. be.

[0054] "Framework" or "FR" residues are those residues other than the HVR residues as defined herein. These are variable domain residues.

[0055] The term "chimeric" antibody refers to an antibody in which portions of the heavy and / or light chains are derived from a particular source or The remaining heavy and / or light chains are identical to or derived from the same species and are derived from a different source. or refers to an antibody that is identical to or derived from a species.

[0056] A "humanized" antibody is an antibody that contains minimal sequence derived from a non-human antibody. Humanized antibodies are antibodies in which the hypervariable region residues of the recipient antibody are modified to confer the desired antibody specificity, affinity, and non-human species such as camels, mice, rats, rabbits, or non-human primates that have the ability to Residues from the hypervariable regions of human immunoglobulin (receptor antibody) are substituted by those from the hypervariable regions of the donor antibody. In certain embodiments, a "humanized" antibody is a non-human (e.g., lactic) antibody. (d) refers to a chimeric antibody containing amino acid residues derived from CDR and amino acid residues derived from human FR. In some instances, framework region (FR) residues of the human immunoglobulin may be similar to those of the corresponding Furthermore, humanized antibodies have the same structure as the recipient antibody, but in which the amino acid sequence is replaced by non-human residues. They may include residues that are not found in the donor antibody. Such modifications may improve antibody performance. Generally, humanized antibodies have at least one, and typically two, amino acid residues. and all or substantially all of the hypervariable loops of , corresponding to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 ( 1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Stru See ct. Biol. 2:593-596 (1992).

[0057] An "affinity matured" antibody has a higher affinity for an antigen than a parent antibody that does not possess such modifications. one or more alterations in one or more CDRs thereof that result in improved affinity of the antibody In some embodiments, the affinity matured antibody has nanomolar binding to the target antigen. Affinity matured antibodies have affinity at the picomolar or even picomolar levels. For example, random selection of CDR and / or framework residues can be performed. Mutagenesis can be carried out, for example, as described by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1 994);Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994- 2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0058] "Amino acid sequence identity percentages" with respect to the polypeptide and antibody sequences identified herein "Percent (%)" or "homology" takes into account any conservative substitutions as part of the sequence identity. After aligning the sequences, the amino acid residues that are identical to the amino acid residues of the polypeptides being compared are It is defined as the percentage of amino acid residues in the candidate sequence that are identical. Alignment for the purpose of determining the centroid can be performed using, for example, BLAST, BLAST-2, , ALIGN, or Megalign (DNASTAR) software. Various methods within the skill of the art may be used with available computer software. This can be achieved by the method described above. One skilled in the art will appreciate that the maximum number of sequences to be compared can be achieved by the method described above. Alignment methods, including any algorithms required to achieve alignment of However, the present invention provides a method for determining the appropriate parameters for measuring the concentration of For purposes of this specification, percent amino acid sequence identity values ​​are calculated using the sequence comparison computer program ALIG. The ALIGN-2 sequence comparison computer program is generated using Ge It was created by nentech, Inc. and the source code is available in the user documentation. and filed with the U.S. Copyright Office, Washington, DC 20559, and The ALIGN-2 program is registered under the accession number TXU510087. Publicly available from entech, Inc., South San Francisco, CA The ALIGN-2 program runs on the UNIX operating system. A computer for use with a system, preferably digital UNIX® V4.0D. All sequence comparison parameters are set in the ALIGN-2 program. It is set by the program and should not be changed.

[0059] "specific" for an epitope on a particular polypeptide or a particular polypeptide target The terms "bind" or "specifically bind" or "specific" are used herein. When used, for example, at least about 10 -4 M, instead of at least about 10 -5 M, instead of at least about 10 -6 M, instead of at least about 10 -7 M's , instead at least about 10 -8 M, instead of at least about 10 -9 M's, Instead of at least about 10 -10 M, instead of at least about 10 -11 M's, Instead of at least about 10 -12 K for the target that is equal to or greater than M D With In some embodiments, the term "specific binding" may be used. The molecule binds substantially to any other polypeptide or polypeptide epitope. binds to a specific polypeptide or an epitope on a specific polypeptide without Refers to a bond. D These include ELISA, surface plasmon resonance (SPR), and fluorescence-activated cell selection. Immunoprecipitation assays can be performed using methods known in the art, such as fluorescence in situ hybridization (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be determined by, for example, methods that do not generally have binding activity. It is measured by determining the binding of a molecule relative to the binding of a control molecule, which is a molecule of similar structure. For example, specific binding can be demonstrated by the presence of a control molecule similar to the target, e.g., an excess of a non-standard molecule. In this case, the binding of the labeled target to the probe can be determined by competition with the labeled target. Specific binding is indicated if it is competitively inhibited by excess unlabeled target.

[0060] As used herein, "treatment" or "treating" refers to a condition that results in a beneficial effect, including a clinical outcome. For the purposes of this application, a beneficial or desired clinical result is a method for achieving a beneficial or desired outcome. The outcomes may include, but are not limited to, one or more of the following: Alleviation of one or more symptoms resulting from the disease, reduction in the extent of the disease, stabilization of the disease (e.g., prevention or delay of disease progression, prevention or delay of disease spread (e.g., metastasis), prevention of disease recurrence, Preventing or delaying the onset of a disease, delaying or slowing the progression of a disease, improving the disease state, or providing disease remission (Partial or total) reduction in the dose of one or more other drugs required to treat the disease , delayed disease progression, increased or improved quality of life, increased weight gain, and / or survival "Treatment" also includes reduction in the pathological consequences of cancer (e.g., tumor volume, etc.). The methods provided herein may include any one or more of these aspects of treatment. is intended.

[0061] An "effective amount" of a CD8 binding agent or composition as disclosed herein is defined as any amount specifically described. for the purposes described, e.g., in vivo CD8 + To perform T cell imaging An "effective amount" is an amount that is sufficient for the purpose stated (e.g., in vivo). CD8 in + This can be determined by known methods for measuring the activity of the antibody, such as by imaging of T cells.

[0062] The term "therapeutically effective amount" refers to, for example, a therapeutically effective amount of a compound that is effective to treat a disease or condition in a subject (e.g., a mammal such as a human). or disorders, e.g., immunotherapeutic agents (immunotherapy agents as described elsewhere herein) effective to "treat" a disease or disorder. In the case of cancer, a therapeutically effective amount of immunotherapy is used. Immunotherapeutics, cell therapy, or cancer vaccines can reduce the number of cancer cells and It can reduce the size or weight of cancer cells, inhibiting their invasion into peripheral organs (e.g., If necessary, the tumor metastasis can be slowed to some extent, and preferably stopped), and tumor metastasis can be inhibited (e.g., In some cases, the tumor growth may be slowed to some extent, and preferably stopped, and the tumor growth may be inhibited to some extent. and / or to some extent one or more of the symptoms associated with cancer Immunotherapeutic agents, cell therapies, or cancer vaccines can reduce the risk of cancer by targeting existing cancer cells. cytostatic and in that they can prevent the growth of and / or kill In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In another embodiment, a therapeutically effective amount is an amount that prolongs survival of a patient. A therapeutically effective amount is an amount that improves progression-free survival in a patient.

[0063] An "individual" or "subject" is a mammal, including but not limited to: , domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject Human.

[0064] As used herein, "responsiveness" refers to the ability of a subject to respond to a therapeutic agent (e.g., an immunotherapeutic agent). A favorable response occurs during or after treatment. An example of a response is the improvement of tumor progression in a subject during or after treatment with a therapeutic agent (e.g., an immunotherapeutic agent). An undesirable example is the inhibition of growth of the cells, which is caused by treatment with a therapeutic agent (e.g., an immunotherapeutic agent). Continued or accelerated growth of the subject's tumor during or after treatment be.

[0065] As used herein, "monitoring disease progression" refers to monitoring a subject (e.g., cancer, subjects diagnosed with an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) Whether the patient's symptoms worsened, stabilized, or improved (i.e., became less severe) refers to the evaluation at successive time intervals to determine whether a Monitoring cancer progression means, in certain cases, monitoring tumor weight or size (tumor regression or tumor growth), time to progression, survival, length of progression-free survival, overall Response rate, duration of response, quality of life, expression and / or activity of disease markers (e.g., certain expression of specific genes and / or proteins), or other criteria known in the art. For example, monitoring changes in the amount of and disease monitoring in patients with cancer, including measuring response to treatment by imaging techniques. Additional techniques for monitoring progress can be used.

[0066] As used herein, "monitoring treatment progress" refers to monitoring the progress of a subject (e.g., cancer, (subjects diagnosed with an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) Whether disease symptoms worsened, stabilized, or improved (i.e., or less severe) after treatment (e.g., treatment with immunotherapy agents). ) or at successive time intervals after treatment. For example, Treatment progress of subjects who have received or are receiving immunotherapeutic agents is monitored by monitoring disease progression. The same criteria used to determine the effectiveness of the product can be used to monitor the product.

[0067] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means " means a substance that is not biologically or otherwise undesirable, e.g., a substance that is without causing any significant undesirable biological effects or and can be administered to a patient without interacting in a harmful manner with any of the other components of the composition. The pharmaceutical composition can be administered by incorporation into the pharmaceutical composition. preferably meets the required standards of toxicological and manufacturing testing, and and / or the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration ) is included.

[0068] As used herein, "in conjunction with" refers to the use of a second agent, such as an immunotherapeutic agent or Administration of a CD8 binding agent, e.g., as described herein, relative to administration of another diagnostic imaging agent For example, the timing of administering a CD8 binding agent described herein in conjunction with an immunotherapeutic agent. Administering the immunotherapeutic agent refers to administering the immunotherapeutic agent before, after, or after the administration of the immunotherapeutic agent. and administering a CD8 binding agent concurrently with or simultaneously with the administration of an immunotherapeutic agent. Before or after administration of a CD8 binding agent and an immunotherapeutic agent, Additional agents may be administered. Additionally or alternatively, sequential administration of CD8 binding agents Other agents may be administered between the administration of the antibody and the immunotherapeutic agent.

[0069] The term "detecting" refers to determining the presence or absence of a substance or The term "CD8" is intended to include quantifying the amount of CD8+ or CD8+ antigens present in a patient's blood. The term refers to the use of the materials, compositions, and methods of this application for qualitative and quantitative determinations. In general, the particular technique used for detection is not critical to the practice of the methods of the present application. For example, "detecting" according to the methods described herein can include detecting the presence of a CD8 polypeptide. or even including observing the absence or alteration of levels of CD8 polypeptide. In some embodiments, "detecting" refers to measuring wild-type CD8 levels (e.g., mRNA levels). The detecting step may include detecting the nucleotide sequence (a level or a polypeptide level). is any value between 10% and 90% or any value between 30% and 60% compared to the control. This includes quantifying the value of a value or a change (increase or decrease) in a value greater than 100%. The detection may be performed by increasing the number of times from 2 to 10 (including 2 and 10) or more. This may include quantifying any value greater than, for example, a 100-fold change.

[0070] The word "label" as used herein refers to a label directly attached to an antibody (e.g., a VHH). A label refers to a detectable compound or composition that is conjugated to or indirectly conjugated with a label. may itself be detectable (e.g., radioisotope-labeled or fluorescently labeled), Or in the case of an enzyme label, catalyzing a detectable chemical alteration of a substrate compound or composition. This can be done.

[0071] References herein to "about" values ​​or parameters are understood by those of ordinary skill in the art. The term "about" as used herein refers to a value or parameter within the normal error range for each such value. Reference to a parameter includes aspects that are directed to that value or parameter itself (and explanations). For example, a statement referring to "about X" includes a description of "X."

[0072] Aspects and embodiments of the present application "comprising" or "including" aspects and embodiments. "consisting of" and "consisting essentially of" It is understood to include the following.

[0073] As used in this specification and the appended claims, the singular forms "a" and "one" are used interchangeably. "an" and "the" indicate that the situation is clearly different. Unless otherwise specified, it includes multiple referents.

[0074] The term "and / or" and phrases such as "A and / or B" are used herein to refer to When used, it includes both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" means "A, B, and / or or C," when used herein, refers to the following embodiments: A, B, and C. ;A, B, or C;A or C;A or B;B or C;A and C;A and B; It is intended to encompass each of B and C; A (alone); B (alone); and C (alone). will be done.

[0075] Certain features of the invention are, for clarity, described in the context of separate embodiments. It is understood that they may also be provided in combination in a single embodiment. For clarity, various features of the invention that are described in the context of a single embodiment will now be described separately. or any suitable subcombination thereof. All combinations of the embodiments relating to the method of use of Each and every combination is treated as if individually and explicitly disclosed herein. and is disclosed herein.

[0076] CD8-binding agents Functional characteristics The CD8 binding agents provided herein may comprise a VHH domain (e.g., a camelid or The VHH comprises a humanized VHH and has one or more of the following properties: (a) CD8 binding activity The agent has a K of about 1 nM or lower. D specifically binds to human CD8; (b ) CD8 binding agents have a β-glucosidase activity of about 0.002 / sec or lower (e.g., about 0.00 18 / sec or approximately 0.00085 / sec) off (c) binding to human CD8; CD8 binding agents have a K of about 1 nM or lower D Binds to cynomolgus monkey CD8 (d) the CD8 binding agent has a cytotoxicity of about 0.004 / sec or less (e.g., For example, k of approximately 0.0037 / sec or approximately 0.0019 / sec off to cynomolgus monkey CD8 (e) the CD8 binding agent binds to CD8 + Inhibits or stimulates T cell activation (f) the CD8 binding agent does not bind to CD8 + does not induce T-cell proliferation; and (g) The CD8 binding agent binds to CD4 + In some embodiments, V The HH domain has one or more characteristics of the CD8 binding agents described herein. In some embodiments, a labeled VHH domain (i.e., conjugated to a detectable label) may be used. The VHH domains (associated with the VHH domain) may be used in combination with one or more of the features of the CD8 binding agents described herein. It has quality.

[0077] The CD8 binding agents described herein bind to CD8 with high affinity and specificity. In some embodiments, the CD8 binding agent may bind any value or range between these values. Contains approximately 1nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0 .15nM, 0.1nM, 0.05nM, 0.02nM, 0.01nM, 0.001nM , or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D Binds to human CD8 In some embodiments, the CD8 binding agent has any value or range between these values. Including about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15nM, 0.1nM, 0.05nM, 0.02nM, 0.01nM, 0.001n M, or lower (e.g., 10 -9 M or lower, e.g., 10 - 9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D In rhesus monkey CD8 In some embodiments, the CD8 binding agent binds to any value between these values. or ranges including approximately 1nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0. 2nM, 0.15nM, 0.1nM, 0.05nM, 0.02nM, 0.01nM, 0. 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g. Ba10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D crabeater In some embodiments, the CD8 binding agent binds to monkey CD8. about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0 .25nM, 0.2nM, 0.15nM, 0.1nM, 0.05nM, 0.02nM, 0 0.01nM, 0.001nM, or lower (e.g., 10 -9 M or it lower than, for example, 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's )K D (b) binds to human CD8 at about 1, including any value or range therebetween; nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM , 0.1nM, 0.05nM, 0.02nM, 0.01nM, 0.001nM, or lower than (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 - 13 M's or 10 -10 M~10 -12 M's)K D binds to rhesus monkey CD8; and (c) about 1 nM, 0.5 nM, 0.4 nM, including any value or range therebetween. nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM, 0.1nM, 0.05n M, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 1 0 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -1 0 M~10 -12 M's)K D In some embodiments, C D8 binding agents have a K of about 150 pM or lower D binds to human CD8 at C D8 binding agents have a K of about 350 pM or lower D cynomolgus monkey CD8 In some embodiments, the CD8 binding agent has a K D Human CD8 and CD8 binding agents have a K of approximately 344 pM. D binds to cynomolgus monkey CD8 In some embodiments, the CD8 binding agent has a K of about 50 pM or lower. D in Binds to human CD8, and CD8 binding agents have a K of about 150 pM or lower D in In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8. K of M D and the CD8 binding agent binds to human CD8 with a K of approximately 137 pM. D crabeater In some embodiments, the CD8 is CD8α. In some embodiments, CD8 is a CD8α / CD8α homodimer. , a CD8α / CD8β heterodimer.

[0078] In some embodiments, the CD8 binding agent may have any value or range between these values. Including, approximately 0.01 / sec, 0.005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, 0.0002 / sec, 0. 0001 / sec, or lower (e.g., 10 -2 / sec or lower For example, 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off Dehi In some embodiments, the CD8 binding agent may be anywhere between these values. Any value or range, including approximately 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.00 1 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0 0.015 / sec, 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 - 4 ~10 -3 / sec)k off In some embodiments, C D8 binding agents may have a D8 activity of about 0.01 / sec, ... .005 / sec, 0.002 / sec, 0.001 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec seconds, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off Crab-eating macaques In some embodiments, the CD8 binding agent (a) binds to CD8. Any value or range of about 0.01 / sec, 0.005 / sec, 0.004 / sec, 0. 003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, 0.0002 / sec, 0.0001 / sec, or lower (e.g., 10 -2 / second or lower, e.g., 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 - 3 / sec)k off (b) any value or range between these values. Including, approximately 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.00 05 / s, 0.004 / s, 0.003 / s, 0.002 / s, 0.0015 / s, 0. 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / second or Lower than that, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec No. off (c) any value or range therebetween. Including, approximately 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.00 05 / s, 0.004 / s, 0.003 / s, 0.002 / s, 0.0015 / s, 0. 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / second or Lower than that, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec No. off In some embodiments, the CD8 binding activity The agent has a K of about 0.002 / sec or less. off binds to human CD8 at The combined agent has a K of about 0.004 / sec or less. off to cynomolgus monkey CD8 In some embodiments, the CD8 binding agent has a K of about 0.0018 / sec. off and the CD8 binding agent binds to human CD8 with a K of about 0.0037 / sec. off Dekaniku In some embodiments, the CD8 binding agent binds to monkey CD8. K for seconds off and CD8-binding agents bind to human CD8 with a K of approximately 0.002 / sec. of f In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 at about 0 K at .00085 / sec off and the CD8 binding agent binds to human CD8 at about 0.00 19 / sec K off In some embodiments, CD8 binds to cynomolgus monkey CD8 at C In some embodiments, the CD8 is a CD8α / CD8α homodimer. In some embodiments, the CD8 is a CD8α / CD8β heterodimer.

[0079] In some embodiments, the CD8 binding agent may have any value or range between these values. about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or longer (e.g., a CD8 binding half-life of at least 15 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to 2 hours It binds to human CD8 with a short life (e.g., in an in vitro binding assay). In embodiments, the CD8 binding agent has a saturation of about 3 to 100 saturates, including any value or range therebetween. 0 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or longer (e.g., at least 1 5 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to 2 hours) In some embodiments, the CD8 binding agent binds to ghesal CD8. Approximately 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or any value or range of longer (e.g., at least 15 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to It binds to cynomolgus monkey CD8 with a CD8 binding half-life of 2 hours.

[0080] The present invention relates to human CD8, rhesus CD8, and / or cynomolgus CD8. The K value of the CD8 binding agent provided in the document D and k off These include, but are not limited to: For example, ELISA, fluorescence-activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA) ), and by any method known in the art, including surface plasmon resonance (SPR). In some embodiments, human CD8, rhesus CD8, and and / or the K of the CD8 binding agents provided herein for cynomolgus monkey CD8. D and / or k off is determined by SPR. The K of the provided CD8 binding agents D and / or k off is CD8α / CD8β- Determined by surface plasmon resonance (SPR) using Fc fusion proteins as reagents In some embodiments, the CD8α / CD8β-Fc fusion protein is a single-arm human In some embodiments, the CD8α / human CD8β-Fc fusion protein The CD8β-Fc fusion protein is a single-arm cynomolgus CD8α / cynomolgus CD8 In some embodiments, the single-arm CD8α / CD8β-Fc fusion protein is a CD8α / CD8β-Fc fusion protein. The Fc fusion protein contains human CD8α and Fc fused to one polypeptide chain of Fc. and human CD8β. In some embodiments, the single-arm CD8α The CD8β / CD8β-Fc fusion protein is a CD8β-Fc fusion protein that is fused to one polypeptide chain of Fc. It contains a single polypeptide chain containing cynomolgus monkey CD8α and cynomolgus monkey CD8β. In embodiments, the antibody is directed against human CD8, rhesus monkey CD8, and / or cynomolgus monkey CD8. The K of the CD8 binding agents provided herein D is determined by FACS. Exemplary human, rhesus, and cynomolgus CD8α amino acid sequences are shown in FIG. are.

[0081] In some embodiments, the CD8 binding agents provided herein bind to mouse CD8. In some embodiments, the CD8 binding agent is , does not bind (e.g., does not specifically bind) to rat CD8. D8 binding agents are identified as D8-binding agents that bind to mouse C It does not bind (eg, does not specifically bind) to either D8 or rat CD8.

[0082] The properties of the CD8 binding agents described herein can be determined by well-known methods, for example, as described in the Examples below. In some embodiments, CD8 + T Cell proliferation was measured using peripheral blood mononuclear cells (PBMCs) and the CD8 binding activity provided herein. In some embodiments, the CD8 + T cell increase The proliferation was performed using PBMCs, anti-CD3 antibodies, anti-CD28 antibodies, and the CD8 antibodies provided herein. In some embodiments, CD8 + T cell proliferation is stimulated by Staphylococcus enterotoxin B (SEB) In vitro in the presence of PBMCs and a CD8 binding agent provided herein. In some embodiments, CD8 + T cell proliferation is driven by the CEF peptide pool In the presence of stimulated PBMCs and a CD8 binding agent provided herein In some embodiments, CD8 + T cell proliferation is associated with lipopolysaccharide PBMCs stimulated with lipopolysaccharide (LPS) and the CD8-binding agents provided herein. In some embodiments, the in vitro assay is performed in the presence of The assay is performed using 10% FBS as the medium. The in vitro assay was performed using 10% autologous donor plasma as the medium, and the donor blood Plasma and PBMCs are obtained from the same donor.

[0083] In some embodiments, the CD8 binding agents provided herein bind to human CD4 + T thin In some embodiments, the vesicles provided herein do not bind (e.g., do not specifically bind). The CD8-binding agent is a human CD3 - Does not bind to cells (e.g., specifically binds to In some embodiments, the CD8 binding agents provided herein bind to human CD4 + T also has CD3 - It does not bind to cells or anything (e.g., it does not specifically bind). In embodiments, the antibodies provided herein are directed against human CD4+ T cells or human CD3− cells. The lack of specific binding by the CD8 binding agents described herein may be due to, as discussed in the Examples, Detection is by fluorescence activated cell sorting (FACS).

[0084] Exemplary CD8 binding agents having one or more of the above functional attributes are provided herein. In some embodiments, agents (including anti-CD8 antibodies and antibody fragments thereof) are provided. Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser4 8, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gl It specifically binds to the human CD8α epitope containing y95, Asp96, and Thr97. CD8 binding agents are provided that comprise a VHH domain having the sequence According to number 13. Also, Arg25, Lys42, Gln44, Val45, Leu46 , Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg Human CD8α epitope containing 93, Leu94, Gly95, Asp96, and Thr97 In some embodiments, a nucleotide sequence is provided, where the amino acid numbering is according to SEQ ID NO: 13. The amino acid residues of the human CD8α epitope bind to human CD8α and one or more amino acid residues of the VHH domain in the crystal structure of the agent or the VHH domain Furthermore, the CD8 binding agents described herein (e.g., antibodies Anti-C antibody that competitively binds to the same human CD8α epitope as one of the CD8 VHHs D8 antibodies are provided.

[0085] In some embodiments, the CD8 binding agents provided herein are specific for human CD8. In some embodiments, the VHH domains provided herein specifically bind to the Camelidae VHH domains. The CD8 binding agent comprises a humanized VHH domain that specifically binds to human CD8.

[0086] In some embodiments, the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 is VHH domains containing at least one, two, or three CDRs of the amino acid sequence CD8 binding agents comprising agonists are provided.

[0087] In some embodiments, (a) the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9; and (c) a CDR2 as set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. At least one, two, or three CDRs selected from CDR3 containing an amino acid sequence CD8 binding agents are provided that comprise a VHH domain that comprises an R.

[0088] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and SEQ ID NO: 10, SEQ ID NO: No. 11, or a VHH domain comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. CD8 binding agents are provided.

[0089] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 1 are 3. A CD8 binding agent comprising a VHH domain comprising:

[0090] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO:2 are 3. A CD8 binding agent comprising a VHH domain comprising:

[0091] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 3 are 3. A CD8 binding agent comprising a VHH domain comprising:

[0092] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 4 are 3. A CD8 binding agent comprising a VHH domain comprising:

[0093] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:6, CDR2 comprises the amino acid sequence of SEQ ID NO:8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10. CD8 binding agents comprising the domain are provided.

[0094] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 9 and CDR3 comprising the amino acid sequence of SEQ ID NO: 11. CD8 binding agents comprising the domain are provided.

[0095] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 7, CDR2 comprises the amino acid sequence of SEQ ID NO: 9 and CDR3 comprising the amino acid sequence of SEQ ID NO: 11. CD8 binding agents comprising the domain are provided.

[0096] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 9 and CDR3 comprising the amino acid sequence of SEQ ID NO: 12. CD8 binding agents comprising the domain are provided.

[0097] Exemplary CDR sequences are shown in Figure 1 and Table 1 below.

[0098] [Table 1]

[0099] In some embodiments, the CD8 binding agent comprises a VHH domain comprising L49A, In this case, the numbering is according to Kabat numbering. An example of an L49A mutation is SEQ ID NO: 2 in FIG. In some embodiments, the L49A mutation is shown in Figures 1-4. This allows for the purification of the CD8 binding agent used. In some embodiments, the L49A mutation The difference is that the yield of the CD8 binding agent is increased by at least about 2-fold, 5-fold, 10-fold, or more. also increases significantly.

[0100] In some embodiments, the CD8 binding agent reduces the immunogenicity of the VHH domain For example, a CD8 binding agent may be administered to a subject receiving the agent, e.g., to induce a CD8 binding reaction against a pre-existing anti-VHH antibody. VHH domains containing one or more framework mutations that reduce binding of therapeutic agents In some embodiments, the CD8 binding agent comprises a V89T substitution, a T110Q substitution, S112Q substitution, and one or more amino acids selected from the group consisting of A114 addition Some embodiments include VHH domains containing acid modifications, in which case the numbering is according to the Kabat numbering system. In embodiments, the VHH domain comprises a V89T substitution, a T110Q substitution, a S112Q substitution, and and A114 addition, in which case the numbering is according to Kabat numbering. Examples of such mutations are shown in SEQ ID NOS: 2-4 in FIG. 1. In some embodiments, framework mutations The difference reduces the immunogenicity of the CD8 binding agent by at least about 2-fold, 10-fold, or 10-fold. Reduce by a factor of 0, 1000, or more.

[0101] In some embodiments, the CD8 binding agent comprises a VH having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD8 binding agent comprises an H domain. In some embodiments, the CD8 binding agent comprises a VHH domain having the sequence In some embodiments, the VHH domain comprises a CD8-binding VHH domain having the amino acid sequence of SEQ ID NO: 3. The therapeutic agent comprises a VHH domain having the amino acid sequence of SEQ ID NO:4.

[0102] Exemplary VHH sequences are shown in FIG.

[0103] In some embodiments, the CD8 binding agents provided herein are renally cleared. In some embodiments, the CD8 binding agents provided herein are cleared by the renal system. be largely removed.

[0104] In some embodiments, an anti-CD8 antibody is provided. Anti-CD8 heavy chain antibodies comprising any one of the VHH domains described above are provided. In some embodiments, the anti-CD8 heavy chain antibody comprises an Fc region, such as a camelid or human Fc region. In embodiments, the anti-CD8 heavy chain antibody is an IgG1, IgG2, IgG3, or IgG In some embodiments, the antibodies provided herein comprise the Fc of CD8 antibodies contain Fc variants that retain some, but not all, effector functions, Thus, the anti-CD8 antibodies provided herein are useful in treating rheumatoid arthritis, as the in vivo half-life of the antibody is not important. However, certain effector functions (such as complement and ADCC) are dispensable or This makes them desirable candidates for harmful applications.

[0105] In some embodiments, an anti-CD8 antibody, such as an anti-CD8 single domain antibody or an anti-CD8 VHH. Antibody fragments are provided.

[0106] In some embodiments, the CD8 binding agent does not comprise an Fc region.

[0107] In some embodiments, the CD8 binding agents provided herein comprise one or more Suitable moieties for derivatization of antibodies include, but are not limited to, non-proteinaceous moieties. However, water-soluble polymers are also included. Non-limiting examples of water-soluble polymers include including but not limited to polyethylene glycol (PEG), ethylene glycol / propylene Glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol Cholesterol, Polyvinylpyrrolidone, Poly-1,3-dioxolane, Poly-1,3,6-trimethylsilyl Oxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymer or and dextran or poly(n-vinylpyrrolidone or a random copolymer). Polyethylene glycol, propylene glycol homopolymer, polypropylene Prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyethylene Examples include polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is stable in water, so The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and may include more than one polymer. If polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization may vary. The specific properties or functions of the antibody that are sought to be improved, including but not limited to, antibody derivatives, are defined. The decision should be based on considerations including whether the product will be used in therapy under certain conditions. can.

[0108] In some embodiments, the CD8 binding agent is a non-tandem compound that increases the serum half-life of the agent. In some embodiments, the CD8 binding agent does not comprise a polyethylene glycol moiety. It does not contain soluble polymers such as polyethylene glycol (PEG).

[0109] b. Mutants and Modifications In some embodiments, the CD8 binding agents described herein (e.g., anti-CD8 antibodies) Amino acid sequence variants of the CD8 binding agent are contemplated. It may be desirable to improve the activity or other biological properties of the CD8 binding agent. Amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein. Such modifications can be prepared by adding methylaminopropyl ... The amino acid sequence of a CD8 binding agent (including one or more CDRs and / or deletions and / or deletions of residues within the VHH domain (e.g., in framework sequences or VHH domains) or insertions and / or substitutions. Any combination of deletions, insertions, and substitutions may be used, provided that the fragment contains a sequence (as described elsewhere in this document). The final construct can be arrived at by

[0110] "CD8 binding agent variant" refers to a polypeptide, e.g., a CD8 binding agent variant, as described herein. CD8 binding agents with desired properties may be selected from the VH of a CD8 binding agent described herein. Such a VHH includes a VHH having at least about 80% amino acid sequence identity with the CD8 binding domain. Synthetic agent variants include, for example, variants in which one or more amino acid residues are present in the VHH domain. Examples include agents that are added to or deleted from the VHH domain. The CD8 binding agent variants may be a CD8 binding agent described herein and at least one about 80% amino acid sequence identity, alternatively at least about 85%, 90%, 95%, 96 %, 97%, 98%, or 99% amino acid sequence identity. Optionally, the variant CD8 binding agent is a CD8 binding agent sequence provided herein. Not more than one conservative amino acid substitution compared to the sequence, instead, the CD about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less compared to the 8-binding agent sequence The amino acid sequence will have any of the following conservative amino acid substitutions:

[0111] In some embodiments, the polypeptides have one or more amino acid substitutions, insertions, and / or deletions. CD8 binding agent variants are provided that exhibit the following properties: Conservative substitutions include HVRs and FRs. Conservative substitutions are listed under the heading "conservative substitutions" in Table 2. More substantial modifications are provided under the heading "Exemplary Substitutions" in Table 2. and as further described below with respect to amino acid side chain classes. Substitutions can be introduced into the antibody of interest to improve the product's ability to retain / improve the desired activity, e.g., antigen binding, immunoglobulin E, or IgG. It is possible to screen for decreased virulence or improved ADCC or CDC. Cut.

[0112] [Table 2]

[0113] Substantial modification of the biological properties of the CD8 binding agent variants can be achieved by (a) modifying the polypeptide in the substituted region; Maintenance of the structure of the peptide backbone, e.g., as a sheet or helix conformation (b) maintaining the charge or hydrophobicity of the molecule at the target site; or (c) the bulkiness of the side chains. This can be achieved by selecting substitutions that have significantly different effects on maintenance. The amino acids can be grouped according to the similarity of their side chain properties (AL Lehn inger, Biochemistry second ed., pp.73-75, Worth Publishers, New York (1975)) : (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro( P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Ty r(Y), Asn(N), Gln(Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0114] Instead, naturally occurring residues are grouped based on common side chain properties. This can be done. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (б) Aromatic: Trp, Tyr, Phe.

[0115] Non-conservative substitutions involve exchanging a member of one of these classes for another. This will result in...

[0116] In some embodiments, the CD8 binding agents provided herein comprise the amino acid sequence of SEQ ID NO: 1. The amino acid sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, VHH domains containing amino acid sequences with 7%, 98%, 99%, or 100% sequence identity In some embodiments, the CD8 binding agents provided herein comprise a domain having the sequence The amino acid sequence of number 2 and at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the CD8-binding domains provided herein comprise a VHH domain comprising The agent has a sequence identical to the amino acid sequence of SEQ ID NO: 3, with a sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 111 4%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the VHH domain comprises a C amino acid sequence as provided herein. The D8 binding agent has at least 90%, 91%, 92%, or 100% affinity to the amino acid sequence of SEQ ID NO:4. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the VHH domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 9 VHH sequences with 9% identity have substitutions (e.g., conservative substitutions), insertions, etc. compared to the reference sequence. CD8 binding agents that contain the sequence, or contain deletions, but contain the sequence, may bind to CD8 (e.g., human retains the ability to bind to human CD8, rhesus monkey CD8, and / or cynomolgus monkey CD8 In some embodiments, the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 A total of 1 to 10 amino acids are substituted, inserted, and / or deleted in In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., the FR ) occurs in the sequence , SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 include.

[0117] One type of substitutional variant is a substitution of one of the amino acids of a parent antibody (e.g., a llama VHH or a humanized VHH). Generally, the target gene is selected for further study and involves substituting one or more hypervariable region residues. The resulting variants selected may have certain biological properties (e.g., affinity) compared to the parent antibody. and / or the parent antibody Exemplary substitution variants are those that substantially retain certain biological properties of the parent. Affinity matured antibodies can be produced by phage cloning, e.g., as described herein. They can be conveniently generated using display-based affinity maturation techniques. In other words, one or more HVR residues are mutated and the mutant antibody is displayed on phage. and screened for specific biological activity (e.g., binding affinity).

[0118] Alterations (e.g., substitutions) can be made to the HVRs to, for example, improve antibody affinity. Such modifications occur in HVR "hotspots," i.e., regions that are highly expressed during the somatic maturation process. Residues encoded by codons that frequently mutate (e.g., Chowdhury, Methods M ol. Biol. 207:179-196 (2008)), and / or for the SDR (a-CDR) The resulting mutant VHHs are tested for binding affinity. Affinity maturation by constructing and reselecting from rallies has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al, ed., Human Press, Totow a, NJ, (2001)). In some embodiments, affinity maturation is carried out using various methods (e.g., For example, error-prone PCR, chain shuffling, or oligonucleotide-directed mutations Diversity is introduced into the variable genes selected for maturation by either heterogeneity (heterogenesis) or by cloning. A secondary library is then generated. The library is then screened to identify the desired Another method for introducing diversity is to identify antibody variants with desired affinities. HVR designation methods include randomizing multiple HVR residues (e.g., 4–6 residues at a time). HVR residues involved in antigen binding can be identified by, for example, alanine scanning mutagenesis or can be specifically identified using modeling. CDR3 is specifically targeted. This is often the case.

[0119] In some embodiments, substitutions, insertions, or deletions are made such that such alterations are related to the target site of the target gene, e.g., a target gene for CD8. The ability of the CD8 binding agent to bind to the nucleotides present within one or more of the CDRs is not substantially reduced. For example, conservative modifications (e.g., those described herein) that do not substantially reduce binding affinity may be made. Conservative substitutions (such as those provided in Table 1) may be made in the CDRs. The variant V provided above may be outside the "hot spot" or SDR of R. In some embodiments of the HH sequence, each HVR is unaltered, or has one, two, or or contains three or fewer amino acid substitutions.

[0120] Useful for identifying antibody residues or regions that can be targeted for mutagenesis. The method was "Alanis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method is called "scanning mutagenesis." or groups of target residues (e.g., loaded groups such as Arg, Asp, His, Lys, and Glu) Identify neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity. Additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted as candidates for substitution or may be excluded. The mutants may be screened to determine whether they contain the desired properties. can be done.

[0121] Amino acid sequence insertions can range from one residue to 100 or more residues. Amino and / or carboxyl terminal fusions ranging in length from and intrasequence insertions of single or multiple amino acid residues. Examples of insertional variants of antibody molecules include antibodies with an N-terminal methionyl residue. The antibody may contain an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of an antibody. Examples of such a method include fusing the antibody to the N-terminus or C-terminus of the antibody.

[0122] c. An immunoconjugate containing a detectable label In some embodiments, the CD8 binding agent is conjugated to a detectable label. Immunoglobulins comprising any one of the anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein. The term "label" or "detectable label" refers to a conjugate of a cell, tissue, or organ. and diagnosis and detection of the location and / or amount of target molecules (such as CD8) in organs, etc. or refers to atoms, molecules, or compounds useful for visualization / imaging. Detectable labels that can be used include, but are not limited to, radioactive radioactive substances (e.g., radioisotopes, radionuclides, radioactive labels, or radioactive tracers) , dyes (e.g., indocyanine green (ICG)), contrast agents, fluorescent compounds, or or molecules, bioluminescent compounds or molecules, enzymes, and imaging agents (e.g., paramagnetic ions) In addition, some nanoparticles, such as quantum dots and metal nanoparticles, can be used for detection. The compounds may be suitable for use as pharmaceutical agents.

[0123] Radioactive substances that can be used as detectable labels in accordance with embodiments herein These include, but are not limited to: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 5 2 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 7 7 As, 86 Y, 89 Sr, 89 Zr, 90 Y, 90 Nb, 94 Tc, 99 Tc, 99 m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154~ 158 Gd, 161 Tb, 166 Dy, 169 Er, 175 Lu, 177 Lu, 186 R e. 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 21 1 Pb, 212Bi, 212 Pb, 213 Bi, 223 Ra, and 225 Ac is mentioned Exemplary paramagnetic ionic substances that can be used as detectable labels include: These include, but are not limited to, transition metals and lanthanide metals (e.g., those with atomic numbers 6-9, ions of metals 21-29, 42-44, or 57-71. The elements are Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, and Pm. , Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu ions are listed. can be done.

[0124] When the detectable label is a radioactive metal or a paramagnetic ion, in some embodiments, the label These molecules have one or more chelating groups attached to their long tails for binding to these ions. The long tail may be reacted with a reagent having a long tail, such as polylysine, polysaccharide, or the like. may have attached a chelate or chelating group (i.e., for binding to ions) polymers such as other derivatized or derivatizable chains having pendant groups. Examples of chelating groups that can be used in accordance with embodiments herein include These include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTA), Acetic acid (DTPA), DOTA, NOIA, NOGADA, NETA, NODA, NOTA , deferoxamine (DfO), DFO * (i.e., DFO-star), DFO-sk Aramids, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, Polyoximes, and similar groups. Chelates minimize loss of immunoreactivity. suppresses adhesion, allowing molecules to form bonds while minimizing aggregation and / or internal cross-linking group linked to an anti-CD8 antibody (e.g., an anti-CD8 VHH) provided herein. It can be complexed with non-radioactive metals (e.g., manganese, iron, and gadolinium). When the same chelate is used in conjunction with a CD8 binding agent described herein, magnetic Useful for magnetic resonance imaging (MRI). NOIA, NOGADA, DOTA, NODA, N Macrocyclic chelates such as, but not limited to, OTA and TETA are suitable for use in, for example, with various metals and radioactive metals, including radionuclides of sodium, yttrium, and copper. Used in radionuclides, such as radium-223 (RAIT) for radioactive iodine treatment. Other cyclic chelators, such as macrocyclic polyethers, are used, which are important for stable binding. In some embodiments, for use in positron emission tomography (PET) analysis, Using the chelating moiety, aluminum- 18 PET imaging agents such as F complexes are described herein. The aluminum- 18 The F complex has the formula (I) It can be conjugated to the VHH domain via

[0125] [ka] For example, U.S. Patent Application Publication No. 20180273441 and Cleeren F. et al. See Nature Protocols 13, 2330-2347 (2018).

[0126] In some embodiments,18 Conjugated to a radionuclide label such as F CD8 binding agents comprising any one of the anti-CD8 VHH domains described are provided. In some embodiments, the VHH domain is conjugated to a label via a chelating moiety. In some embodiments, the chelating moiety is attached to the VHH domain via a lysine residue. In some embodiments, the radionuclide label is included in a metal complex. In some embodiments, the radionuclide label forms a complex with a metal, and the complex is a chelate. In some embodiments, the CD8 binding agent is chelated by a moiety. 18 F mark Antibodies conjugated to chelating moieties that chelate complexes containing cations and aluminum In some embodiments, the chelating moiety comprises a compound of formula (I): It is a thing.

[0127] In some embodiments, the compound of formula (I) 18 F]-aluminum fluoride complex and any one of the anti-CD8 VHH domains described herein conjugated to CD8 binding agents are provided.

[0128] In some embodiments, the compound of formula (I) 18 F]-aluminum fluoride complex a CD8 binding agent comprising a VHH domain conjugated to a The CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 10. a CD8-binding agent comprising a CDR2 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 3. nothing.

[0129] In some embodiments, the compound of formula (I) 18 F]-aluminum fluoride complex a CD8 binding agent comprising a VHH domain conjugated to a The CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 10. a CD8 binding agent comprising a CDR2 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 4. nothing.

[0130] Use as a detectable label in accordance with embodiments of the methods and compositions herein Exemplary contrast agents that can be used include, but are not limited to: Contains: barium, diatrizoate, ethiodized oil, citric acid Gallium, iocarminic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid , Ioglamid, Iohexyl, Iopamidol, Iopanoic acid, Ioprosemic acid, Io Cefamic acid, iocelic acid, iosuramide meglumine, iosemetic acid , iotasul, iotetolic acid, iothalamic acid, iotroxic acid, ioxagalic acid, iox Sotrizoic acid, ipodate, meglumine, metrizamide, metrizoate ate), propriodone, thallium chloride, or a combination thereof.

[0131] Bioactive compounds that can be used as detectable labels in accordance with the methods and compositions herein. Luminescent and fluorescent compounds or molecules and dyes include, but are not limited to: For example, fluorescein, fluorescein isothiocyanate (F ITC), OREGON GREEN (trademark), Rhodamine, Texas Red, IRDy e800CW, ALEXA FLUOR® 647, Tetrarhodamine isothiocyanate Fluorescent markers (e.g., green fluorescent) such as trimethylsilyl citrate (TRITC), Cy3, and Cy5 proteins (e.g., GFP and phycoerythrin), activated by tumor-associated proteases Self-quenching fluorescent compounds, enzymes (e.g., luciferase, horseradish peroxidase) that are quenched by fluorescing. enzymes, and alkaline phosphatases), nanoparticles, biotin, digoxigenin, Or a combination thereof.

[0132] Enzymes that can be used as detectable labels in accordance with the methods and compositions herein Examples of the enzyme include, but are not limited to, horseradish peroxidase, alkali, etc. Phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase beta-lactamase, beta-glucoronidase, or beta-lactamase. The enzyme can be used in combination with a chromogen, fluorogenic compound, or luminescent compound to produce a detectable A signal can be generated.

[0133] In some embodiments, the CD8 binding agents provided herein are nanoparticles, i.e. , conjugated to microscopic particles whose size is measured in nanometers. For example, a nanoparticle is a particle having at least one dimension less than about 100 nm. They are small enough that they scatter visible light rather than absorbing it, making them detectable. For example, gold nanoparticles have significant visible light extinction properties and can be used in solution. appears deep red to black. As a result, the nanoparticles conjugated to the compounds provided herein The CD8 binding agents can be used for in vivo imaging of T cells in a subject. At the lower end of this size range, nanoparticles are often referred to as clusters. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles) ) are formed. Nanospheres, nanorods, and nanocups are grown in the shape These are just a few examples. Semiconductor quantum dots and nanocrystals are examples of additional types of nanoparticles. Such nanoscale particles can be used in combination with anti-CD8 antibodies (e.g., anti-CD8 antibodies) provided herein. When conjugated to a nucleotide sequence (e.g., a CD8 VHH), T cells are transduced in vivo as described herein. It can be used as an imaging agent to detect vo.

[0134] The conjugate of the antibody and label was N-succinimidyl-3-(2-pyridyldithio) E) Propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)silane Monohexane-1-carboxylate (SMCC), iminothiolane (IT), imidene Bifunctional derivatives of esters (e.g., dimethyl adipimidate HCl), activated esters (e.g., suberyl esters), disuccinimidyl phosphate, etc.), aldehydes (glutaraldehyde, etc.), bis-azides compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives Conductors (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates esters (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1, Various bifunctional protein caps, such as 5-difluoro-2,4-dinitrobenzene, For example, ricin immunotoxins can be prepared using coupling agents. al, Science 238:1098 (1987). Isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See brochure 94 / 11026. The linker is used to inhibit the release of the cytotoxic drug in the cell. It may also be a "cleavable linker" that facilitates release. For example, an acid labile linker, Protease-sensitive linker, photolabile linker, dimethyl linker, or disulfide containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208 The immunoconjugates of the present specification can be , including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC , MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, ST Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SI AB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimide crosslinker reagents including, but not limited to, benzoyl-(4-vinylsulfone)benzoate Conjugates prepared using PEG-400 are commercially available (e.g., Pie rce Biotechnology, Inc., Rockford, IL, USA In some embodiments, the CD8 binding agents provided herein are and a linker that is a benzoxamine compound (see, e.g., Vugts et al. (2017) Eur J Nucl Med M ol Imaging. 44:286-295 and Rudd et al. (2016) Chem Commun. 52: 11859-12000. In some embodiments, the CD8 binding agents provided herein are N-succinimide. In some embodiments, the linker comprises a dihydro-desferrioxamine (DFO) linker. The CD8 binding agents provided are desferrioxamine compounds (e.g., N-succinimide). radionuclides (such as, but not limited to, 8 9 Zr, 124 I, or 18 Anti-CD8 VHH conjugated to IgG (including F) In some embodiments, the label may be, for example, an enzyme, such as a sortase or transglucanase. Conjugated to anti-CD8 VHH domains in a site-specific manner using tamminase It has been done.

[0135] In some embodiments, the CD8 binding agents provided herein are labeled with a detectable label. Directly coupled (i.e., without a linker) anti-CD8 VHH domains Includes.

[0136] Methods for producing CD8 binding agents Also described herein are methods for producing anti-CD8 antibodies (e.g., anti-CD8 VHHs). and methods for producing labeled CD8 binding agents. Methods for producing 8-binding agents are provided.

[0137] The anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein are described, for example, in U.S. Pat. and produced using recombinant methods and compositions as described in US Pat. No. 6,015,695. In some embodiments, an anti-CD8 antibody described herein (e.g., an anti-CD8 Isolated nucleic acids encoding anti-CD8 VHs are provided. Such nucleic acids may be used in combination with anti-CD8 VHs. In some embodiments, the anti-CD8 V domain may encode an amino acid sequence comprising an H domain. An isolated nucleic acid encoding an HH domain, comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a nucleic acid sequence encoding SEQ ID NO: 4, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 1 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity Nucleic acids are provided that include sequences having the following structure:

[0138] In some embodiments, a vector (e.g., an expression vector) comprising a nucleic acid described herein. In some embodiments, a host cell comprising such a nucleic acid or vector is provided. In some embodiments, the host cell is a eukaryotic organism, such as a Chinese hamster. - ovarian (CHO) cells, Expi293 cells, or lymphoid cells (e.g., Y0, NS In some embodiments, the host cell is a prokaryotic cell, e.g., a colony-stimulating cell. In some embodiments, the antibody is an anti-CD8 antibody (e.g., an anti-CD8 V HH), comprising nucleic acid encoding an antibody as provided above. Culturing the host cells under conditions suitable for expression of the antibody, and optionally (or the host cell culture medium).

[0139] A method for preparing a labeled CD8 binding agent, comprising: conjugated to any one of the anti-CD8 antibodies (e.g., anti-CD8 VHHs) described providing a conjugate comprising an anti-CD8 antibody and a chelating moiety; Induce Gate, 18 The labeled CD8-binding antibody is contacted with an aluminum fluoride complex containing F. The method further comprises providing an agent, wherein the chelating moiety is a compound of formula (I). In some embodiments, the chelating moiety binds to a lysine residue of the anti-CD8 antibody. In some embodiments, the conjugate is conjugated to one or more antibodies. In some embodiments, one of the aluminum fluoride complexes is contacted with the aluminum fluoride complex in the presence of an oxidizing compound. or the plurality of antioxidant compounds are methionine and / or N-acetyl-tryptophan In some embodiments, the conjugate comprises methionine and N-acetyl-trimethylsilyl In some embodiments, the method comprises contacting the compound with an aluminum fluoride complex in the presence of hydroxybenzoate. The labeled CD8 binding agent is added to a reaction mixture containing the conjugate and aluminum fluoride. In some embodiments, the desalting column is , histidine, methionine, N-acetyltryptophan, and / or sucrose In some embodiments, the desalting column is equilibrated with a buffer containing histidine, methionine, The column is equilibrated with a buffer containing tryptophan, N-acetyltryptophan, and sucrose.

[0140] To recombinantly produce an anti-CD8 antibody (e.g., an anti-CD8 VHH), for example, the above-described Nucleic acids encoding the antibodies are isolated and further cloned and / or purified in host cells. The nucleic acid is inserted into a vector for expression. Such nucleic acids can be expressed using conventional procedures (e.g., as an antibody). oligonucleotides capable of specifically binding to genes encoding the heavy and light chains of the human body; These can be easily isolated and sequenced (by using a nucleotide probe).

[0141] Suitable host cells for cloning or expressing antibody-encoding vectors include those described herein. For example, antibodies may be produced from prokaryotic or eukaryotic cells, particularly those described in the literature. and if Fc effector functions are not required, they can be produced in bacteria. For expression of antibody fragments and polypeptides in Escherichia coli, see, e.g., U.S. Patent No. 5,648,222. 37, 5,789,199, and 5,840,523 See Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed. , Humana Press, Totowa, NJ, 2003), pp. 245-254. Expression of antibody fragments in E. coli has been described. After expression, the antibody is transferred to bacterial cell plates. The soluble fraction can be isolated from the yeast and further purified.

[0142] In addition to prokaryotes, other cloning or expression hosts for antibody-encoding vectors include In some cases, the glycosylation pathway has been "humanized" to allow for partially or fully human glycosylation patterns. and filamentous fungi or yeast, including fungal or yeast strains that result in the production of antibodies having the Eukaryotic microorganisms are preferred. Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li See, e.g., Wang et al., Nat. Biotech. 24: 210-215 (2006).

[0143] Suitable host cells for the expression of glycosylated antibodies also include multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, transfection of Spodoptera frugiperda cells The application features numerous baculovirus strains that can be used in conjunction with insect cells. It is set forth.

[0144] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,929,139 and 5,930,142. Specification No. 59,177, Specification No. 6,040,498, Specification No. 6,420,548 , 7,125,978, and 6,417,429 (Transgen PLANHBODIES™ technology for producing antibodies in nicked plants is described. Please refer to the

[0145] Vertebrate cells can also be used as hosts, for example, cells grown in suspension. Mammalian cell lines that have been adapted for this purpose may be useful. Other useful mammalian host cell lines include: Examples are the SV40(COS-7) transformed monkey kidney CV1 line; the human embryonic kidney line (e.g., 293 or 29 as described in Graham et al., J. Gen Virol. 36:59 (1977) 3 cells; baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., Mather, TM4 cells as described in Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells ( W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); For example, TR as described in Mather et al., Annals NY. Acad. Sci. 383:44-68 (1982) Other useful mammalian host cell lines include I cells; MRC5 cells; and FS4 cells. DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1 Chinese hamster ovary (CHO) cells, including 980); and Y0, NS0, and Certain mammalian host cell lines suitable for antibody production include myeloma cell lines such as Sp2 / 0 and Sp2 / 0. For a review of main cell lines, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0146] CD8 binding agents are used to + Detect, localize, and / or image cells How to make it As used herein, the CD8 binding agents described herein (e.g., anti-CD8 antibodies, or Use one of the following: an immunoconjugate containing an anti-CD8 antibody and a detectable label And CD8 + Methods for detecting, localizing, and / or imaging cells are provided. In some embodiments, the method comprises detecting CD4 in an in vitro or ex vivo sample. In some embodiments, the method comprises detecting the presence of 8. For example, this method includes adding a CD8 binding agent to the ex vivo sample. These include, but are not limited to, Western blot, immunohistochemical analysis, and ELISA assay. Such methods, including those involving the use of CD8-binding agents, may optionally include administering the CD8-binding agents in vitro. Alternatively, the method may include adding the ex vivo sample to the sample and then washing the sample. In some embodiments, the step of detecting binding of the CD8 binding agent to CD8 comprises detecting binding of the CD8 binding agent to an anti-CD8 VHH In some embodiments, the method comprises detecting a label attached to the anti-C domain. a step of applying a second agent comprising the detectable label herein that binds to the CD8:CD8 complex; the step of detecting binding of the CD8 binding agent to CD8 includes a step of detecting binding of a second agent to the CD8 binding agent. Those skilled in the art will recognize that the secondary agent binds to CD8 and detects the detectable label of the secondary agent. The binding of the CD8-binding agent to the CD8-binding agent does not compete with the CD8-binding agent or inhibits the binding of the CD8-binding agent to the CD8-binding agent. It will be easy to see that they are not competing.

[0147] In some embodiments, the methods include detecting, localizing, or detecting the presence of CD8 in vivo. In some embodiments, the method comprises the step of detecting or imaging CD8 as described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal, e.g., a rat, a mouse, a guinea pig, , hamsters, rabbits, dogs, cats, cows, horses, goats, sheep, donkeys, pigs, monkeys, etc. In some embodiments, the non-human primate is a macaque monkey, a rhesus monkey, or other non-human primate. In some embodiments, the CD8 binding agent is a human macaque or a cynomolgus macaque. In some embodiments, the CD8 The binding agent is administered to the subject by infusion (e.g., intravenous infusion). In some embodiments, the CD8 binding agent is administered intravenously or intraperitoneally. In some embodiments, the method comprises administering the CD8 binding antibody to the subject by injection, such as by subcutaneous injection. administering a CD8 binding agent to a subject and analyzing (i.e., CD8 binding agent and CD8 The method includes removing a sample from the subject for detection of binding to the antibody.

[0148] In some embodiments, CD8 + The detection, localization, and / or imaging of cells can be performed using, for example, For example, in vivo, using techniques described in more detail elsewhere herein. It will be carried out.

[0149] In some embodiments, detecting the presence of CD8 in vivo comprises detecting CD8 (CD8 + Some embodiments involve localizing a target cell or organ relative to a tissue. In some embodiments, the method includes detecting CD8 in an organ or tissue of a subject, e.g., in a diseased tissue. + The number of cells In some embodiments, the subject has cancer and determines an in vivo The step of detecting the presence of CD8 comprises detecting CD8 + This involves localizing cells to the tumor. In some embodiments, CD8 + The cells are CD8 + T cells, e.g. tumor-infiltrating CD8 + In some embodiments, the method comprises detecting CD8 T cells in a tumor of a subject with cancer. + In some embodiments, the method comprises determining the number of T cells in a subject having cancer. CD8 in elephant tumors + determining the number of T cells at multiple consecutive time points. .

[0150] In some embodiments, CD8 +The cells were found to be between these values ​​after administration of the CD8 binding agent. Approximately 6 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 1 hour, 3 hours, including any value or range of Within 0 minutes or less (for example, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours) Within about a day or less, such as within 2 hours or about 2-4 hours It can be detected, localized, or imaged in vivo.

[0151] In some embodiments, CD8 + The cells may be dosimetrically assayed using any of the methods described herein. 1, 2, 3, 4, 5 or more times per year without exceeding the guidelines , used one or more times to detect, localize, or image in vivo. In some embodiments, the method comprises administering the CD8 binding agent to the patient about 7 days after the first administration of the CD8 binding agent. It can be repeated after 6, 5, 4, 3, 2, 1, or less days. Cut.

[0152] In some embodiments, the labeled CD8 binding agents may be labeled with one or more CD8 binding agents for multiplexed imaging. In some embodiments, one or more A number of additional imaging agents may be administered within a short period of time after administration of the labeled CD8 binding agent, e.g., As soon as the radiation from one imaging agent has decayed, e.g., about 48 hours, 36 hours, 24 hours, Within 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour or less In some embodiments, the longevity Unlike bioimaging reagents, the labeled CD8 binding agents described herein do not induce additional immune responses. For characterization, CD8 imaging and standard of care PET imaging (e.g., FDG-PET) ) or in combination with novel molecular imaging (e.g., CD4, Granzyme B, PSMA) In some embodiments, the method includes imaging using a labeled CD8 binding agent. Another imaging scan (e.g., PET such as FDG-PET, SP) should be performed within approximately 48 hours of the initial scan. The method further comprises the step of performing a CT scan or scintigraphy scan.

[0153] In some embodiments, this method can be used to calculate the variance of a given value, including any value or range between these values. Yes, at least about 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or more For extended periods, such as longer than + Detect and localize cells The immunogenicity of the CD8 binding agents described herein can be measured or imaged. Therefore, the use of CD8-binding agents for in vivo imaging and CD8 detection is limited. Repeated and long-term use is possible.

[0154] In some embodiments, the method provides a method for detecting CD8 in vivo in a manner that is between these values. about 1 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20 nM, including any value or range of about 1 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20 nM, M, 25 nM, 30 nM, 40 nM, or 50 nM (e.g., at least about 50 nM, For example, about 1 nM to about 50 nM, or about 1 nM to about 30 nM) of CD8. In embodiments, the method comprises determining the relationship between the signal from the label and the CD8 level in vivo. In some embodiments, the method comprises: + Tumors (e.g., TA LL-1) xenograft model, at least about 2, 3, 4, 5, 6, 7, 8, 9, 1 Having a tumor:blood ratio of 0, 11, 12, 13, 14, 15, 20, or higher .

[0155] Techniques for in vivo detection of CD8 In some embodiments, the CD8 binding agent and CD8 (e.g., CD8 + Cells, e.g. CD8 + In vivo binding to T cells was measured using immuno-PET (positron emission tomography) , SPECT (single photon emission computed tomography), NMR (nuclear magnetic resonance) Also known as MRI (Magnetic Resonance Imaging), Near Infrared (NIR), or Cerenkov Luminescence Imaging detected by at least one of the following imaging methods: Cerenkov luminescence imaging (CLI) In some embodiments, the binding of the CD8 binding agent to CD8 is via two or more In some embodiments, the CD8 binding agent and the CD8 binding agent are detected by many forms of imaging. Binding to 8 is detected by near infrared (NIR) and / or CLI. In this embodiment, the binding of the CD8 binding agent to CD8 is detected by immunoSPECT and / or NIR In some embodiments, the binding of a CD8 binding agent to CD8 is detected by: It is detected by immunoSPECT and computed tomography.

[0156] ImmunoPET is 18 F, 64 CU, 68 Ga, 76 Br, 86 Y, 89 Zr, and 1 24 Antibodies labeled with positron-emitting radionuclides, such as I (anti-CD8 antibodies provided herein) The method is based on the simultaneous detection of the CD8 antibody and its fragments. Examples of the radioactive nuclides include, but are not limited to,18 F, 64 Cu, 68 Ga, 76 Br, 86 Y, 88 Y, 89 Zr, 99m Tc, 111 In, 177 Lu, 123 I, 1 24 I, 125 I, and 131 The emitted positrons are Depending on the energy and density of the surroundings, they can travel distances of up to several millimeters ( See, for example, Table 2 in Guus et al. (2007) The Oncologist, 12: 1379-1389. After losing energy, the positron joins with the electron in a process known as annihilation, This produces two photons, each with an energy of 511 keV. are simultaneously released in opposite directions. The distribution can be monitored by detecting the annihilation photon pairs with a PET camera. The ET camera consists of a ring of detectors placed around the patient's body. It is recorded by detectors on both sides of the body within a very short time interval (usually 5-15 nanoseconds). If so, it is assumed that the annihilation event occurred somewhere along the line between the two detectors. The position of the radioactive source (radiolabeled antibody) is determined by calculating the intersection of all the lines. Regarding quantification, PET can provide reliable information if appropriate corrections are made. (Verel et al. (2005) J Nucl Med, 46 suppl 1: 164S-171S) For additional details on immunoPET, see, for example, van Dongen et al. (2007) The Onc ologist, 12(12): 1379-1389;Reddy et al. (2010) Semin Nucl Med. 40(3): 182-189; Boerman et al. (2011) J. Nucl Med. 52(8): 1171-1172;Santangelo et al. (2015) Na ture Methods, 12: 427-432.

[0157] ImmunoSPECT imaging involves the use of antibodies labeled with gamma-emitting radionuclides (as provided herein). The antibody or fragment thereof is typically injected into the patient's bloodstream. Examples of gamma-emitting radionuclides include, but are not limited to: for example, 67 Ga, 99m Tc, 111 In, 123 I, 131 I, 153 Sm, or 186 Next, a gamma camera is used to capture multiple 2D images from multiple angles. A computer is then used to perform a tomographic reconstruction algorithm on the multiple projections to obtain a 3D dataset. This dataset can then be manipulated to produce other It displays thin slices along any selected axis of the body, similar to those obtained from tomography techniques. To acquire SPECT images, a gamma camera is rotated around the patient. Projections are taken at defined points during rotation, usually every 3-6 degrees. To obtain the best possible reconstruction, a full 360 degree rotation is used. The time it takes to complete the process can vary, but 15-20 seconds is typical. The total scan time is 15-20 minutes. In some cases, SPECT gamma scanners , and built to work with conventional CT scanners using image coregistration. This allows tumors that can be observed by SPECT scintigraphy to be detected. or tissue localization, but accurately localizing it relative to other anatomical structures Additional details regarding immunoSPECT can be found, for example, in Laverman et al. 015) J Nucl Med, 56(5): 778-783;Lutje el al. (2014) Cancer Res, 74(21): 6216-62 23; and Muselaers et al. (2013) Eur Urology 64(4): 1101-1106. can.

[0158] In vivo MRI (Magnetic Resonance Imaging), also known as NMR (Nuclear Magnetic Resonance) The principle of the ion beam irradiation is to ionize the protons and neutrons (most commonly The mechanism of nuclear motion is based on manipulating the magnetic properties of atoms (e.g., those found in hydrogen atoms). The subject's body is placed in the magnetic field of the MRI scanner. The magnetic moments of these nuclei then align with the direction of the magnetic field. When a radio frequency (RF) pulse is applied to the body, nuclei are excited and form low-energy spin states. A transition occurs between the nucleus and the high-energy spin state. When the RF pulse ends, the nucleus It returns to equilibrium (a process called relaxation) and releases the excess absorbed energy, resulting in RF This signal is detected by the RF coil of the scanner, which then This is used to produce detailed images of body tissues. MRI contrast agents are used This improves the contrast of this image and therefore the visibility of certain body structures. Examples of labels that can be detected by MRI include, but are not limited to, , superparamagnetic iron oxide (Molday ION Rhodamine-B Carboxyl iron oxide nanoparticles, such as 19 F-based probes, paramagnetic metals (e.g., Gadolinium nium, manganese, manganese oxide, dysprosium), (U)SPIO, PARA(CE ST), DIA (CEST), and PFC. Additional information regarding the use of labeled antibodies and / or MRI-detectable labels can be found, e.g., in Srivastava (2015) Dis Model Mech. 8(4): 323-336;Zhou et al. (2013) Wiley In terdiscip Rev Nanomed Nanobiotechnol. 5(1): 1-18;Sohn et al. (2015) Nanomedicin e. 11(1): 127-135;Bates et al. (2014) PloS ONE 9(5): e97220;Zhu et al. (2015) Int. J. Mol. Sci. 16: 9573-9587; and Zhang et al. (2014) Int J. Medicine. 9: 3 This is discussed in 3-41.

[0159] NIR imaging utilizes the deep photon penetration power of near-infrared light into biological tissue to a depth of <1 cm. This provides imaging of the intrinsic and / or extrinsic contrast of NIR fluorescent light within this field. Optical imaging involves the use of exogenous contrast agents labeled with fluorescent light emitting between 700 and 900 nm. The focus is on antibody detection. Exemplary fluorescence imaging systems are described in detail elsewhere. (De Grand et al. (2003) Technol Cancer Res Treat, 2:553-62; Nakayama et al. (2002) Mol Imaging, 1: 365-77;Ntziachristos et al. (2003) Eur Radiol, 13 :195-208;Tanaka et al. (2006) Ann Surg Oncol, 13:1671-81;Themelis et al. (2009 ) J Biomed Opt, 4:064012; and Troyan et al. (2009) Ann Surg Oncol, 16:2943-52 ) Briefly, a typical fluorescence imaging system excites fluorophores in turbid media. Spectrally resolved light sources (filtered broadband light sources, light emitting diodes [LEDs]) The light emitted from this fluorophore is then , taking special care to filter out the strong excitation light, The image is captured by a CCD camera. Examples of infrared dyes include, but are not limited to, Tr acy652, Tracy645, rhodamine dyes, cyanine dyes, Cy7, Cy7.5 , ALEXA FLUOR (registered trademark), CYDYE (registered trademark), IRDYE (registered trademark) These include the near-infrared (NIR) laser, DyLight, and ATTO. Cell and tissue imaging at near-infrared (NIR) wavelengths is difficult due to the low absorption of biological molecules in this region. This is advantageous for in vivo imaging. Labeled antibodies for in vivo NIR imaging and further details on the use of detectable labels for in vivo NIR imaging. Killers et al. (2017) Mol Pharmaceuticals 14(5): 1623-1633;Hilderbrand et a l. (2010) Curr Opin Chem Biol 14(1): 71-79;Hong et al. (2017) Nat Biomed Eng 1, 0010 DOI: 10.1038 / s41551-016-0010;Pansare et al. (2012) Chem Mater. 24(5): 812 -827;Hickson (2009) Urol Oncol Semin Orig Invest. 27: 295-297;Zhang et al. (20 12) Curr Protoc Cytom. Chapter 12: Unit 12.7;Quek et al. (2012) Nanomaterials. 2: 92-112; Luker et al. (2008) J Nucl Med 49: 1-4; and Liu et al. (2016) NPG A Sia Materials. 8, e295.

[0160] Cerenkov luminescence imaging (CLI) is a positron emission tomography (PET) imaging agent (Honmei Analysis based on the detection of optical Cherenkov photons emitted by Other CLI imaging agents include, but are not limited to, Ba, 131 I, 18 F, and 90 Y. Cherenkov radiation is the radiation produced by charged particles A dielectric medium (i.e., a medium that can be polarized by an electric field) is polarized by a force that moves faster than the speed of light in that medium. During propagation, charged particles (positively charged positrons or negatively charged electrons) ) induces local polarization by displacing positive and negative charges on the atoms of the medium See, for example, Figure 1 in Grootendorst et al. (2016) Clin Transl Imaging. 4(5): 353-366. When the particle speed exceeds the speed of light, the polarization becomes asymmetric along the particle's trajectory, At greater distances from the atom, a dipole electric field is created. As the particle passes, the atom's electrons The electron returns to the ground state, whereby the transition energy is released as an optical photon. PET trays are scanned using an ultra-sensitive optical camera such as an electron-coupled device (EMCCD) camera. CLI images can be obtained by detecting the Cherenkov light from the C. LI images can be analyzed semiquantitatively for photon brightness. Both CLI and PET Both techniques measure photons generated by positron-emitting radiopharmaceuticals, so they can be used directly. They are correlated. PET measures annihilation photons, CLI measures Cherenkov photons. Studies have shown that for various radiopharmaceuticals, in vitro, ex vivo, and i It has been shown that there is a strong correlation between CLI and PET in vivo. The feasibility of molecular imaging of living subjects using CLI has been demonstrated. Publications detailing the correlation between CLI and PET include, for example, Xu et al. (2012) J Nucl Med, 53(2):312-317;Liu et al. (2010) PLoS ONE. 5(3):e9470;Zhang et al. (2013) PLoS ONE. 8(4):e62007;Hu et al. (2015) Eur Radiol. 25(6): 1814-1822;Ro bertson et al. (2011) J Nucl Med. 52(11): 1764-1769;Timmermand et al. (2015) J Nucl Med. 56(3):444-449;Cao et al. (2014) Biomed Opt Express. 5(10):3660-3670, and Thorek et al. (2014) J Nucl Med. 55(1):95-98.

[0161] Methods for predicting responsiveness of a subject with cancer to immunotherapy Also provided is a method for predicting the responsiveness of a subject with cancer to treatment with an immunotherapeutic agent. In some embodiments, the method comprises administering a labeled CD8 binding agent; and a labeled CD8-binding agent and CD8 in the tumor tissue of interest. + Detecting binding to T cells wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. In some embodiments, the methods include administering a labeled CD8 binding agent described herein. The CD8 binding agent and the CD8 binding agent of the target tumor tissue were then analyzed. + Binding to T cells was detected detecting binding indicates that the subject is in need of treatment with the immunotherapeutic agent. In some embodiments, the CD8 binding agent is labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 and labeled with a CD8-binding agent. , CD8 in tumor tissue + Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 Anti-CD conjugated to F-label 8 VHH. In some embodiments, the CD8 binding agent is linked via a compound of formula (I) do[ 18 F]-aluminum fluoride complex conjugated to anti-CD8 VHH. In some embodiments, the anti-CD8 VHH comprises a CDR comprising the amino acid sequence of SEQ ID NO: 7. 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. Contains the amino acid sequence.

[0162] In some embodiments, the methods include administering a therapeutically effective amount of an immunotherapeutic agent, a cell therapy, or a cancer vaccine. The vaccine (e.g., a personalized cancer vaccine or "PCV") is administered by injecting a labeled CD8 binding agent into a tumor Tissue CD8 + The method includes administering the antibody to a subject in which binding to T cells has been detected.

[0163] In some embodiments, the CD8 binding agent may be used to monitor a subject's responsiveness to an immunotherapeutic agent. In some embodiments, the method is administered more than once to predict the return. at least about 6 months, 1 year, 2 years, 3 years, 4 years, or any value or range between the values Repeated over a long period of time, such as five, ten, or even longer.

[0164] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is ipilimumab (YERVOY®). In some embodiments, the immune checkpoint inhibitors are therapeutic anti-CTLA-4 antibodies, such as In some embodiments, the therapeutic anti-PD-1 antibody is In some embodiments, the therapeutic anti-PD- One antibody is pembrolizumab (KEYTRUDA®). In this study, the therapeutic anti-PD-1 antibody was pidlizumab.

[0165] In some embodiments, the immune checkpoint inhibitor is a therapeutic anti-PD-L1 antibody. In some embodiments, the therapeutic anti-PD-L1 antibody is BMS-936559. In this embodiment, the therapeutic anti-PD-L1 antibody is avelumab (BANVENCIO®). In some embodiments, the therapeutic anti-PD-L1 antibody is durvalumab (IM In some embodiments, the therapeutic anti-PD-L1 antibody is Tezolizumab (TECENTRIQ®).

[0166] For further details regarding therapeutic immune checkpoint inhibitors, see, for example, Byun et al. 017) Nat Rev Endocrinol. 13: 195-207;La-Beck el al. (2015) Pharmacotherapy. 35( 10): 963-976;Buchbinder et al. (2016) Am J Clin Oncol. 39(1): 98-106;Michot et al. al. (2016) Eur J Cancer. 54: 139-148, and Topalian et al. (2016) Nat Rev Canc er. 16: 275-287.

[0167] In some embodiments, the immune checkpoint inhibitor is administered in combination with one or more additional therapeutic agents. In some embodiments, one or Immune checkpoint inhibitors administered to a subject in combination with multiple additional therapeutic agents (e.g., chemotherapy agents) PD-L1 inhibitors are anti-PD-L1 antibodies (e.g., atezolizumab). Examples of chemotherapy agents include Examples include: Erlotinib (TARCEVA®, Genentech / OSI Pharmaceuticals) m.), bortezomib (VELCADE®, Millennium Pharmaceuticals m.), disulfiram, epigallocatechin gallate, salinosporamide A, carfil Zomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca eca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), Imachi Nib mesylate (GLEEVEC®, Novartis), finasunate (fina sunate) (VATALANIB®, Novartis), oxaliplatin ( ELOXATIN (registered trademark, Sanofi), 5-FU (5-fluorouracil), Leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth ), lapatinib (TYKERB®, GSK572016, GlaxoSmi th Kline), lonafamib (SCH 66336), Sorafenib nib (NEXAVAR®, Bayer Labs), gefitinib (IRES SA®, AstraZeneca), AG1478, thiotepa and CYTO Alkylating agents such as XAN® cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan ; benzodopa, carboquone, meturedopa, and uredopa Aziridines such as (uredopa); Altretamine, triethylenemelamine, triethylenephosphoramide, triethylene Thiophosphoramide, and ethylenimine and methyl melamine, including trimethylmelamine Lamin (methylamelamine); Acetogenins (especially bullatacin and bullatacinone); camptothecins (topotecan and and irinotecan); bryostatin; kallistatin; CC-1065 (including its additives) including zelesin, carzelesin, and biceresin synthetic analogs); cryptophycins ( especially cryptophycin 1 and cryptophycin 8); corticosteroids (prednisolone) cyproterone acetate; finasteride and dutasteroids 5α-reductases containing riboside; vorinostat, romidepsin, panobinostat, Luproic acid, mocetinostat dolastatin; aldesleukin, talc duocalmay synth (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; Pancratistatin; Sarcodictiin; Spongistatin; Chlorambucil, chlomaphazine, chlorophos phamide), estramustine, ifosfamide, mechlorethamine, mechlorethamine oxy Hydrochloride, melphalan, nobuenbikinin, fenesterine, prednimustine, trophoblast nitrogen mustards such as sulfamide and uracil mustard; Carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranitidine nitrosoureas such as ranimnustine; Enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γII and Calicheamicin ω II (Angew Chem. Intl. Ed. Engl. 1994, 33:183-186); dynemicins including phenanthrene A; bisphosphonates such as clodronate; esperamicin; and Neocarzinostatin chromophore and related chromoproteins (enediyne antibiotic chromophores) Which antibiotics, aclacinomycin, actinomycin, anthracycline Isin (authramycin), azaserine, bleomycin, cactinomycin, carabicin ( carabicin), caminomycin, carzinophilin, chromomycin chromomycinis), dactinomycin, daunorubicin, detorubicin, 6-diazo-5- Oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), Morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin Bicine, and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, Marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin Syn, olivomycin, peplomycin, porfiromycin, puromycin, que Ramycin, rhodolubicin, streptonigrin, streptozocin, tubercidin, Benimex, zinostatin, zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as terin, methotrexate, pteropterin, and trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didethiol Pyrimidines such as oxyuridine, doxifluridine, enocitabine, and floxuridine analog; Calsterone, dromostanolone propionate, epithiostanol, mepitiostane androgens such as acetaminophen, testolactone; any antiadrenal medication; folic acid supplements such as frolinic acid; Aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; Amsacrine; Bestravsil; Bisantrene; Edatrexate; Def Defofamine; Demecolcine; Diaziquone; Elfomithin e); Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxy urea; lentinan; lonidainine; maytansine and ansamitocin Maytansinoids such as; mitoguazone; mitoxantrone; mopidamnol ol); nitraerine; pentostatin; phenamt; pirarubicin; Soxantrone; Podophyllic acid; 2-ethylhydrazide; Procarbazine; PSK ( Trademark) Polysaccharide Complex (JHS Natural Products, Oregon; Razoxane; Rhizoxin; Sizofuran; Spirogel Manium;Tenuazonic acid;Triaziquone;2,2',2''-Trichlorotriethyla trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mito Lactol; pipobroman; gacytosine; arabinoside ("Ara-C") cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel) Bristol-Myers Squibb Oncology, Princeton, NY (Jersey), ABRAXANE® (Cremophor-free), Paclitaxel Albumin-engineered nanoparticle formulations of cisplatin of paclitaxel)(American Pharmaceutical Partner s, Schaumburg, Illinois), and TAXOTERE® (docetaxel , doxetaxel (Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; thotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); Novantrone; Teniposide; Edat Rexate; daunomycin; aminopterin; capecitabine (XELODA® )); Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Zif difluoromethylornithine (DMFO); retinoids such as retinoic acid; and the above; Any pharmaceutically acceptable salts, acids, and derivatives.

[0168] Chemotherapeutic agents also include: (i) For example, tamoxifen (NOLVADEX®); tamoxifen citrate raloxifene, droloxifene, iodoxyfene, 4-hydroxybenzoates Droxitamoxifen, Trioxifen, Keoxifen, LY117018, Ona Pristone, and FARESTON® (toremifene citrate) Antiestrogens and selective estrogen receptor modulators (SERs), including SER citrate Antihormones that act to regulate or block hormone action on tumors, such as steroid hormones (M) Agents; (ii) For example, 4(5)-imidazole, aminoglutethimide, MEGASE Trademark) (megestrol acetate), AROMASIN® (exemestane; Pf izer), Formestani, Fadrozole, RIVISOR (registered trademark) (Vorozole), FEMARA® (Letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca) Aromatase inhibitors, which block the enzyme aromatase, which regulates estrogen production in the adrenal glands, Harmful agents; (iii) flutamide, nilutamide, bicalutamide, leuprolide, and goserelin Antiandrogens such as buserelin, triptorelin, and medroxyacetate Progesterone, diethylstilbestrol, Premarin, fluoxymesterone, All trans-retionic acid, fenretinide, and thromboxane Sacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those directed against, for example, PKC-alpha, Ral Genetic analysis of signaling pathways implicated in abnormal cell proliferation, such as f and H-Ras. those that inhibit the expression of offspring; (vii) VEGF expression inhibitors (e.g., ANGIOZYME®) and H ribozymes such as inhibitors of ER2 expression; (viii) Gene therapy vaccines, e.g., ALLOVECTN®, LEU vaccines such as VECTIN® and VAXID®; PROLE UKIN®, rIL-2; LURTOTECAN®, and other topoisomers ABRELIX® rmRH; and (ix) Pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0169] Chemotherapeutic agents also include antibodies such as: alemtuzumab (C ampath), bevacizumab (AVASTIN®, Genentech); Cetuximab (ERBITUX®, Imclone); panitumumab (VEC TIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec, pertuzumab (OMNITARG (Registered Trademark), 2C4, Genentech), trastuzumab (HERCEPTIN (Registered Trademark) (Genentech), tositumomab (Bexxar, Corixia), and and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG (trademark), Wyeth). Therapeutic potential as an agent in combination with the compounds of the present application. Additional humanized monoclonal antibodies include: apolizumab, apolizumab Celizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab Mertansine, cedelizumab, certolizumab pegol, cidfusituzumab umab), cidtuzumab, daclizumab, eculizumab, efalizumab, Epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozo ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab , matuzumab, mepolizumab, motavizumab, natalizumab Mab, nimotuzumab, nolovizumab, numavizumab, Clerizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab (pecf) usituzumab, pectuzumab, pexelizumab, ralivizumab mab), ranibizumab, reslivizumab, reslivizumab, resivizumab (resyvizumab), rovelizumab, ruplizumab, Sibro Sibroutuzumab, siplizumab, sontuzumab, tacatuzumab Tetraxetane (tacatuzumab tetraxetan), tadocizumab (tadocizumab), Talizma tefibazumab, tocilizumab, toralizumab, Tucotuzumab celmoleukin, tucusituzuma b), umavizumab, urtoxazumab, ustekinumab, visilizumab, and and recombinant interleukin-12 p40 protein ABT-874 / J, a fully human full-length IgG1λ antibody 695, Wyeth Research and Abbott Laboratori es).

[0170] Furthermore, chemotherapeutic agents include "EGFR inhibitors," which directly bind to EGFR. specifically binds to or otherwise interacts with a protein, preventing or reducing its signaling activity, These compounds are also referred to as "EGFR antagonists." Examples of such agents include: Antibodies and small molecules that bind to EGFR include: Examples include MAb579 (ATCC CRL HB8506), MA b455 (ATCC CRL HB8507), MAb225 (ATCC CRL 85 08), MAb528 (ATCC CRL 8509) (U.S. Patent No. 4,943,533 See Mendelsohn et al., and chimerized 225 (C 225 or cetuximab; ERBUTIX®), and modified human 225 (H225) (International Publication No. 96 / 40210 Pamphlet, Imclone S their variants, such as IMC-11F8, fully human; EGFR-targeting antibody (Imclone); antibody that binds to type II mutant EGFR (US US Patent No. 5,212,290; US Patent No. 5,891,996 humanized and chimeric antibodies that bind to EGFR, as described in Human antibodies that bind to EGFR, such as panitumumab (WO 98 / 50433 Panitumumab) FRET, see Abgenix / Amgen); EMD55900 (Stragliotto et al. l. Eur. J Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), EGFR binding Humanized EGF against EGFR that competes with both EGF and TGF-alpha for binding. GFR antibody (EMD / Merck); human EGFR antibody HuMax-EGFR(G enMab);E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6 .3, and E7.6.3, and are described in U.S. Pat. No. 6,235,883. MDX-447 (Medarex Inc.); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375- 30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents, thus providing immunotherapy. Conjugates can be produced (see, for example, European Patent Application Publication No. 659439). (See the specification, Merck Patent GmbH) as an EGFR antagonist are described in U.S. Patent Nos. 5,616,582; 5,457,105; Specification No. 75,001; Specification No. 5,654,307; Specification No. 5,679,683 ;Specification No. 6,084,095;Specification No. 6,265,410;No. 6,455,53 Specification No. 4; Specification No. 6,521,620; Specification No. 6,596,726; No. 6,7 Specification No. 13,484; Specification No. 5,770,599; Specification No. 6,140,332 ;Specification No. 5,866,572;Specification No. 6,399,602;No. 6,344,45 Specification No. 9; Specification No. 6,602,863; Specification No. 6,391,874; No. 6,3 Nos. 44,455; 5,760,041; 6,002,008; and No. 5,747,498; and the following PCT publication: WO 98 / 1445 Pamphlet No. 1, International Publication No. 98 / 50038 Pamphlet, International Publication No. 99 / 09 016 pamphlet and compounds described in WO 99 / 24037 pamphlet Specific small molecule EGFR antagonists include the following: Examples include: OSI-774 (CP-358774), erlotinib, and TARCEVA. (Registered Trademark) Genentech / OSI Pharmaceuticals; PD1 83805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluoro (4-morpholinyl)propoxy]-6-quinazolidinyl nyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRE SSA® 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6 -(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM1051 80((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca );BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1 -methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diazo Min, Boehringer Ingelheim); PKI-166 ((R)-4-[ 4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidine-6- (R)-6-(4-hydroxyphenyl)-4-[(1-phenyl)-phenol (Nylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynami EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]- 3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butene mid) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer; lapatinib (TYKERB®, GSK572016, or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[ 2-Methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazoline amine Dual EGFR / HER2 tyrosine kinase inhibitors, such as EGFR-1, EGFR-2, HER2-1, and HER2-2.

[0171] Chemotherapeutic agents also include the following: EGFR, as described in the preceding paragraph; "Tyrosine kinase inhibitors," including FR-targeting drugs; available from Takeda Pharmaceutical Co., Ltd. Small molecule HER2 tyrosine kinase inhibitors such as TAK165; CP-724,714, E Oral selective inhibitors of rbB2 receptor tyrosine kinase (Pfizer and OSI);E Preferentially binds to GFR but inhibits both HER2 and EGFR-overexpressing cells Dual HER inhibitors such as EKB-569 (available from Wyeth); lapatinib (GS K572016; available from Glaxo-SmithKline), oral HER2 and and an EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis) pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf inhibits ISIS-1 signaling, available from ISIS Pharmaceuticals Raf-1 inhibitors such as the antisense agent ISIS-5132; imatinib mesylate ( GLEEVEC® (available from GlaxoSmithKline) Non-HER-targeted TK inhibitors; sunitinib (SUTENT®, from Pfizer) multi-targeted tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK2 VEGF such as 22584, available from Novartis / Schering AG Receptor tyrosine kinase inhibitor; MAPK extracellular regulated kinase I inhibitor CI-1040 ( Available from Pharmacia; PD153035, 4-(3-chloroanilino)ky Quinazolines such as nazoline; Pyridopyrimidines; Pyrimidopyrimidines; CGP59326 pyrrolopyrimidines such as CGP60261, CGP62706, and the like; dine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin ( Diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide; nitro Thiophene-containing tyrphostines; PD-0183805 (Wa rner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding HERs) quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (tryphostin) (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca);PTK-787(Novartis / Schering AG);C Pan-HER inhibitors such as I-1033 (Pfizer); Affinitac ( ISIS3521; Isis / Lilly; Imatinib mesylate (GLEEVEC ( PKI166 (Novartis); GW2016 (Glaxo Smith); hKline);CI-1033(Pfizer);EKB-569(Wyeth), Maxinib (Pfizer); ZD6474 (AstraZeneca); PTK-78 7(Novartis / Schering AG);INC-1C11(Imclone ), rapamycin (sirolimus, RAPAMUNE®); or Any of the following patent publications: U.S. Pat. No. 5,804,396 Detailed information: International Publication No. 1999 / 09016 Pamphlet (American Cyana mid); International Publication No. 1998 / 43960 Brochure (American Cya namid); International Publication No. 1997 / 38983 (Warner Lam bert); International Publication No. 1999 / 06378 (Warner Lamb ert); International Publication No. 1999 / 06396 (Warner Lambe rt); International Publication No. 1996 / 30347 (Pfizer, Inc.); Country International Publication No. 1996 / 33978 (Zeneca); International Publication No. 1996 / Pamphlet No. 3397 (Zeneca), and WO 1996 / 33980 Bonus (Zeneca).

[0172] Chemotherapeutic agents include the following: dexamethasone, interferon, ron, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, Alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, aspirin Paraginase, live BCG, bevacizumab, bexarotene, cladribine , clofarabine, darbepoetin alfa, denileukin 2, dexrazoxane, epo Etin alfa, erlotinib, filgrastim, histrelin acetate, ibuprofen Britumomab, interferon alfa-2a, interferon alfa-2b, Nalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nof Nofetumomab, oprelvekin, palifermin, pamidronate, pegasus Demase, pegaspargase, pegfilgrastim, pemetrexed disodium, Plicamycin, porfimer sodium, quinacrine, rasburicase, sargramos Chim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, Valrubicin, zoledronate, and zoledronic acid and their pharmaceutically acceptable salts Salt to be used.

[0173] In addition, chemotherapy drugs include hydrocortisone, hydrocortisone acetate, and cortisone acetate. tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol ol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinonide Lonacetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, phosphate Acid dexamethasone sodium, fluocortolone, hydrocortisone-17-butyrate , hydrocortisone-17-valerate, alclometasone dipropionate (aclometa betamethasone dipropionate), betamethasone valerate, betamethasone dipropionate, prednisolone Clobetasol, Clobetasone-17-butyrate, Clobetasol-17-propionate, Fluocortolone caproate, fluocortolone pivalate, and fluprednide acetate Phenylanine-glutamine-glycine (FEG) and its D-isomer form (fe G) Immunoselective anti-inflammatory drugs such as (IMULAN BioTherapeutics, LLC) Anti-inflammatory peptides (ImSAIDs); azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline omideminocycline), antirheumatic drugs such as sulfasalazine, etanercept (Enb rel), infliximab (Remicade), adalimumab (Humira), Tumor necrosis, such as lutolizumab pegol (Cimzia) and golimumab (Simponi) Interleukin-1 (IL-1) inhibitors, such as tumor necrosis factor alpha (TNFα) blockers and anakinra (Kineret) interleukin-1 (IL-1) blockers, T-cell costimulation blockers such as abatacept (Orencia), Interleukin-6 (IL-6), such as tocilizumab (ACTEMERA®) Interleukin-13 (IL-13) blockers, such as lebrikizumab; Interferon alpha (IFN) blocking agents such as mab (rontalizumab); rhuMAb beta7 integrin blockers, such as beta7; anti-M1 prime; IgE pathway blockers; secreted homotrimeric LT, such as anti-lymphotoxin alpha (LTa) a3 and membrane-bound heterotrimeric LTa1 / β2 blockers; radioisotopes (e.g., At 2 11 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); thioplatin, P S-341, phenylbutyrate, ET-18-OCH3, or farnesyl trans A wide variety of investigational agents, including ferrase inhibitors (L-739749, L-744832); Quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin Polyphenols such as phenols, flavanols, procyanidins, betulinic acid, and their derivatives autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol beta-lapachone; lapachol ; colchicine; betulinic acid; acetylcamptothecin, scopoletin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL ( bexarotene (TARGRETIN®); clodronate (e.g., For example, BONEFOS® or OSTAC®), etidronate ( DIDROCAL®), NE-58095, zoledronic acid / zoledronate ( ZOMETA®), alendronate (FOSAMAX®), Pami Tiludronate (AREDIA®), Tiludronate (SKELID®) ), or bisphosphonates such as risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); THERATOPE® vaccine any vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoric acid) proteasome inhibitors (e.g., PS341); CCI-779 ; Tipifarnib (R11577); Orafenib, ABT510; Obli Bcl-2 inhibitors, such as Mersen sodium (GENASENSE®); Santron; lonafarnib (SCH6636, SARASAR™) and other nesyltransferase inhibitors; and pharmaceutically acceptable salts, acids, or the like of any of the above. or derivatives; as well as cyclophosphamide, doxorubicin, vincristine, and CHOP, an abbreviation for combination therapy with 5-FU and prednisolone; Treatment regimens with oxaliplatin (ELOXATIN™) in combination with thiazolinone A combination of two or more of the above, such as the abbreviation FOLFOX.

[0174] As a chemotherapeutic agent, nonsteroidal anticoagulants with analgesic, antipyretic, and anti-inflammatory effects are also available. NSAIDs include non-selective anti-inflammatory drugs that inhibit the enzyme cyclooxygenase. Specific examples of NSAIDs include: aspartame, Pyrine, ibuprofen, fenoprofen, ketoprofen, flurbiprofen, propionic acid derivatives such as xaprozin, naproxen, indomethacin, thoracic acid, Acetic acid derivatives such as dac, etodolac, and diclofenac, piroxicam, meloxicam, and tetroxin Enolic acids such as noxic acid, droxicam, lornoxicam, and isoxicam olic acid derivatives, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenam Fenamic acid derivatives such as phenamic acid, as well as celecoxib, etoricoxib, lumiracoxib, COX-, such as parecoxib, rofecoxib, rofecoxib, and valdecoxib 2 inhibitors.

[0175] In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered in combination with one of the following chemotherapeutic agents: and administered in combination with one or more of: anti-HER2 antibodies (e.g., trastuzumab (HERCEPTIN®, Genentech) or pertuzumab (PER JETA®, Genentech), PD1 binding antagonists (e.g. , MDX-1106 (nivolumab), MK-3475 (pembrolizumab, lambrolizumab) mab), CT-011 (pidilizumab), or AMP-224), and PD-L2 Binding antagonists.

[0176] In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is a growth inhibitory agent A "growth inhibitory agent," as used herein, is administered in combination with A compound or composition that inhibits cell growth either in vitro or in vivo Exemplary growth inhibitory agents include, for example, (vincristine and vinblastine), taxanes (docetaxel E®, Rhone-Poulenc Rorer), and paclitaxel ( TAXOL®, Bristol-Myers Squibb), and Xorubicin, epirubicin, daunorubicin, etoposide, and bleomycin topoisomerase II inhibitors, such as tamoxifen, prednisone, and dacarbazine , mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and a Such agents that arrest G1, such as DNA alkylating agents like ra-C, also cause S-phase arrest. For more information, see Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineopl astic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995), e.g. 1 It can be found on page 3.

[0177] In some embodiments, the immunotherapeutic agent is a dendritic cell activator or a dendritic cell growth factor. In some embodiments, the immunotherapeutic agent is a vaccine adjuvant. In some embodiments, the immunotherapeutic agent is a T cell stimulator or growth factor. Therapeutic agents act by neutralizing or inhibiting suppressive immune cells, cytokines, and / or enzymes. It is a drug.

[0178] In some embodiments, the method comprises administering to a subject an anti-TIGIT antibody, a TIGIT antagonist, an anti-CS F-1R antibody, anti-CSF-1R antagonist, anti-CEA antibody, anti-CEA antagonist , anti-CTLA4 antibody, CTLA4 antagonist, anti-OX40 antibody, OX40 agonist any anti-PDL1 antibody in combination with one or more chemotherapeutic agents, one or more Any anti-PD1 antibody in combination with a chemotherapeutic agent, and any anti-PD1 antibody in combination with one or more chemotherapeutic agents The method includes administering an immunotherapeutic agent selected from the group consisting of atezolizumab in combination with In some embodiments, the anti-PD1 antibody or anti-PDL1 antibody is administered using TARCEVA (registered trademark). ® (erlotinib), ZELBORAF® (vemurafenib), GAZY VA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (Benib) rafenib) and COTELLIC® (cobimetinib), ALECENSA (Alectinib), KADCYLA (Ado-trastuzumab) HERCEPTIN® (trastuzumab), PERJETA® (registration) (Registered trademark) (pertuzumab), polatuzumab, IFN-alpha, anti-CD40, anti-OX4 0 antibodies (e.g., OX40 agonists), anti-CSF-1R antibodies, anti-CEA antibodies, IDO inhibitors In some embodiments, the compound is combined with one or more of an anti-inflammatory agent, an anti-inflammatory drug, or an anti-TIGIT antibody. The anti-PD-L1 antibody is atezolizumab, which is marketed under the trademark TARCEVA. (erlotinib), ZELBORAF® (vemurafenib), GAZYV A® (obinutuzumab), AVASTIN® (bevacizumab), C OTELLIC® (cobimetinib), ZELBORAF® (Vemula) phenib) and COTELLIC® (cobimetinib), ALECENSA® ®) (alectinib), KADCYLA® (ado-trastuzumab emta) HERCEPTIN® (trastuzumab), PERJETA® trademark) (pertuzumab), polatuzumab, IFN-alpha, anti-CD40, anti-OX40 Antibodies (e.g., OX40 agonists), anti-CSF-1R antibodies, anti-CEA antibodies, IDO inhibitors The compound is combined with one or more of the following antibodies: an anti-CTLA4 antibody, an anti-TIGIT antibody, or an anti-IL-1 antibody. In some embodiments, the immunotherapeutic agent is a cytokine. The antibodies are IL2, genetically modified IL2, IL15, or genetically modified IL15. In embodiments, the immunotherapeutic agent is a dendritic cell activator, such as a dendritic cell growth factor or dendritic cell activator. It is a modulator.

[0179] In some embodiments, the cell therapy is chimeric antigen receptor T-cell (CAR-T) therapy. In some embodiments, the cell therapy is a genetically engineered T cell receptor T cell (TCR-T) therapy. In some embodiments, the cell therapy is a neo-antigen-specific T cell therapy.

[0180] Methods for monitoring the progression of a subject with cancer - Patents.com Provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering to a subject a labeled CD8 binding agent, and At a time point and a second time point, the labeled CD8 binding agent and the CD8 binding agent in the tumor tissue of the subject are measured. + T cells In some embodiments, the method comprises detecting binding to the antibody. administering to the subject an immunotherapeutic agent (e.g., an immunotherapeutic agent described elsewhere herein); and the disease is progressing in the subject. (a) administering a labeled CD8 binding agent to a subject prior to administering an immunotherapeutic agent; D8-binding agents and CD8 in tumor tissues + (b) detecting binding to T cells; (c) administering a labeled CD8 binding agent at a time point after administration of the immunotherapeutic agent; The labeled CD8-binding agent and the CD8 in the tumor tissue were then administered to the subject. + Detecting binding to T cells and (d) measuring CD8 expression in tumor tissue before and after administration of the immunotherapeutic agent. + T cell Measuring the difference in labeling.

[0181] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor is an anti-PD1 antibody (including, but not limited to, the present invention). In some embodiments, the antibody is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody described in the specification). The inhibitor may be an anti-PD-L1 antibody (including, but not limited to, an anti-PD-L1 antibody described herein). In some embodiments, the anti-PD-L1 antibody is atezolizumab. In this embodiment, the anti-PD-L1 antibody (such as atezolizumab) is administered in combination with a second therapeutic agent (such as including, but not limited to, immunotherapeutic and / or chemotherapeutic agents described elsewhere herein) In some embodiments, the second therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody or an anti-PD1 antibody, These include anti-TIGIT antibodies, TIGIT antagonists, anti-CSF-1R antibodies, and anti-CSF- 1R antagonist, anti-CEA antibody, anti-CEA antagonist, anti-OX40 antibody, OX4 0 agonist, anti-CTLA4 antibody, CTLA4 antagonist, TARCEVA (registered trademark) (erlotinib), ZELBORAF® (vemurafenib), GAZYV A® (obinutuzumab), AVASTIN® (bevacizumab), C OTELLIC® (cobimetinib), ZELBORAF® (Vemula) phenib) and COTELLIC® (cobimetinib), ALECENSA® ®) (alectinib), KADCYLA® (ado-trastuzumab emta) HERCEPTIN® (trastuzumab), PERJETA® trademark) (pertuzumab), polatuzumab, IFN-alpha, anti-CD40 agents, or ID The compound is further combined with one or more of the following O inhibitors:

[0182] In some embodiments, the immunotherapeutic agent is a cytokine. The kine is IL2, genetically modified IL2, IL15, or genetically modified IL15.

[0183] In some embodiments, the immunotherapeutic agent is a dendritic cell modulator. In the present case, the immunotherapeutic agent is a dendritic cell activator or a dendritic cell growth factor.

[0184] In some embodiments, the efficacy of the immunotherapeutic agent is assessed by measuring the CD8 + T cells The level of CD8 in the tumor tissue at the first time point was detected. + Compared with T cell levels In some embodiments, disease progression is determined by measuring the C of the tumor tissue at the second time point. D8 + T cell levels were compared with CD8 in tumor tissue at the first time point. + higher than the level of T cells In some embodiments, CD8 in tumor tissue is detected. + The level of T cells is third, In some embodiments, the time points are at least Also 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years In some embodiments, the CD8 + T cell levels are It is detected after administration of immunotherapy agents to patients.

[0185] In some embodiments, the administration of one or more dosing regimens of immunotherapeutic agents to tumor tissue The efficacy is measured by a CD8 binding agent at a first time point and a second time point. In some embodiments, the level of CD8+ T cells in the tumor tissue is determined. is the level (or localization) of CD8+ T cells in tumor tissue after administration of an immunotherapeutic agent to a subject. ) is a measure of CD8 binding activity at a first time point before administration of the immunotherapeutic agent and at a second time point after administration. Determined by comparing the levels of CD8+ T cells in tumor tissue measured by the agent will be done.

[0186] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 1 24 I, 18 F, 68 The tumor tissue is labeled with a CD8-binding agent. Woven CD8 + Binding to T cells is detected by PET or PET / CT. In embodiments, the CD8 binding agent is 18 Anti-CD8 VH conjugated to F-label H. In some embodiments, the CD8 binding agent is administered via a compound of formula (I): 1 8 F]-aluminum fluoride complex conjugated to anti-CD8 VHH. In one embodiment, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, the sequence CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR comprising the amino acid sequence of SEQ ID NO: 11 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 6. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. Includes.

[0187] In some embodiments, the CD8 binding agent is administered to repeatedly monitor the progression of the subject's cancer. In some embodiments, the subject is administered more than once to achieve a range between these values. At least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, inclusive of any value or range It is monitored over a long period of time, such as a year or longer.

[0188] Methods for monitoring the treatment progress of a subject with cancer - Patents.com As used herein, treatment with an immunotherapeutic agent (e.g., an immunotherapeutic agent described elsewhere herein) To monitor the progress of treatment in subjects with cancer who have previously received or are currently receiving Such methods include administering a labeled CD8 binding agent in conjunction with an immunotherapeutic agent. and measuring the level of labeled CD8 binding at the first and second time points. Antibiotics and CD8 in tumor tissue + Detecting binding to T cells. In this embodiment, the labeled CD8 binding agent is administered before the immunotherapeutic agent, and the first time point is the time point at which the labeled CD8 binding agent is administered. the first time point is after administration of the 8-binding agent but before administration of the immunotherapeutic agent, and the second time point is after administration of the immunotherapeutic agent. In some embodiments, the CD8 + T cell levels , a lower value compared to the first time point indicates positive treatment progress (e.g., beneficial or desirable outcome). In some embodiments, the CD8 + T cell A higher level compared to the first time point may indicate a lack of treatment progression (e.g., beneficial or indicates a lack of desired clinical outcome). In some embodiments, the immunotherapeutic agent is a labeled CD8-binding the first time point is after administration of the immunotherapeutic agent and includes labeled CD8 binding; In some embodiments, the second time point is after administration of the combined agent, and the second time point is after the first time point. , CD8 in tumor tissue at the second time point + Lower levels of T cells compared to the first time point A positive result indicates positive therapeutic progress (e.g., a beneficial or desired clinical outcome). Morphologically, CD8 in tumor tissue at the second time point + T cell levels compared to the first time point A higher level indicates a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical results). In some embodiments, the methods involve identifying mechanisms of treatment failure, such as tumor CD8 + Cell loss used to explain whether the loss of efficacy is due to fatigue, fatigue, and / or loss of therapeutic efficacy. In some embodiments, the tumor tissue is CD8 + T cell levels are third, fourth, or fourth In some embodiments, the time point is at least about 1 day, 3 days, or 5 days. , 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or longer intervals than 3 years It is being done.

[0189] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody (e.g., as described elsewhere herein). In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered as a second therapeutic agent. (e.g., as described elsewhere herein) to the subject.

[0190] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 1 24 I, 18 F, 68 The tumor tissue is labeled with a CD8-binding agent. Woven CD8 + Binding to T cells is detected by PET or PET / CT. In embodiments, the CD8 binding agent is 18 Anti-CD8 VH conjugated to F-label H. In some embodiments, the CD8 binding agent is administered via a compound of formula (I): 1 8 F]-aluminum fluoride complex conjugated to anti-CD8 VHH. In one embodiment, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, the sequence CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR comprising the amino acid sequence of SEQ ID NO: 11 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 6. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. Includes.

[0191] In some embodiments, the CD8 binding agent is administered to repeatedly monitor the subject's progress in treatment. In some embodiments, the subject is administered more than once to achieve a range between these values. At least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, inclusive of any value or range It is monitored over a long period of time, such as a year or longer.

[0192] A method for predicting the responsiveness of a subject with cancer to treatment with a cancer vaccine; and methods for monitoring disease progression in subjects with cancer who have been administered a cancer vaccine - Patents.com The present invention provides a method for predicting the responsiveness of a subject with cancer to treatment with a cancer vaccine. In some embodiments, the cancer vaccine is a personalized cancer vaccine. ("PCV"). Exemplary PCVs are described, for example, in Ott et al. (2017) Nature 547, 21 7-221 and Sahin et al. (2017) Nature 547, 222-226. In some embodiments, the method includes administering a labeled CD8 binding agent described herein; and A labeled CD8-binding agent and CD8 in the tumor tissue of interest + Steps to detect binding to T cells The detection of binding indicates that the subject is likely to respond to the cancer vaccine. In embodiments of the method, the method comprises administering a labeled CD8 binding agent described herein. and a labeled CD8 binding agent and CD8 in the tumor tissue of interest. + Detecting binding to T cells detecting binding indicates that the subject is in need of treatment with the cancer vaccine. In some embodiments, the cancer vaccine comprises one or more of the immunoglobulins described herein. It is administered in combination with therapeutic and / or chemotherapeutic agents.

[0193] Also provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering to a subject a CD8 binding agent described herein. and administering the labeled CD8 binding agent to the tumor tissue of interest at a first time point and a second time point. Woven CD8 + In some embodiments, the method comprises detecting binding to a T cell. In some embodiments, the method further comprises administering a therapeutically effective amount of a cancer vaccine. The vaccine is a personalized cancer vaccine (“PCV”).

[0194] As used herein, a cancer vaccine is a cancer vaccine that has previously been or is currently being treated with a cancer vaccine. In some embodiments, methods are provided for monitoring the treatment progress of a subject having The cancer vaccine is a personalized cancer vaccine ("PCV"). In some embodiments, the method (a) administering a labeled CD8-binding agent to a subject before administering a cancer vaccine (e.g., PCV); The labeled CD8-binding agent and the CD8 in the tumor tissue are administered to the subject. + Detecting binding to T cells (b) administering a cancer vaccine (e.g., PCV); (c) administering a cancer vaccine At a time point after administration of a steroid (e.g., PCV), a labeled CD8 binding agent is administered to the subject, and the labeled CD8 binding agent is administered to the subject. CD8-binding agents and CD8 in tumor tissue + detecting binding to T cells; and d) CD8 in tumor tissue before and after administration of a cancer vaccine (e.g., PCV) + T cell In some embodiments, the method comprises measuring the difference in labeling. , e.g., tumor CD8 + Due to cell loss, exhaustion, and / or therapeutic efficacy Used to describe loss or damage.

[0195] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 1 24 I, 18 F, 68 The tumor tissue is labeled with a CD8-binding agent. Woven CD8 + Binding to T cells is detected by PET or PET / CT. In embodiments, the CD8 binding agent is 18 Anti-CD8 VH conjugated to F-label H. In some embodiments, the CD8 binding agent is administered via a compound of formula (I): 1 8 F]-aluminum fluoride complex conjugated to anti-CD8 VHH. In one embodiment, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, the sequence CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR comprising the amino acid sequence of SEQ ID NO: 11 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 6. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. Includes.

[0196] In some embodiments, the CD8 binding agent is administered to a subject in a repeated predictive or monitoring fashion. In some embodiments, the dose is any value between these values. At least approximately 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, including any value or range The method is repeated or the subject is Be monitored.

[0197] A method for predicting the responsiveness of a subject with cancer to treatment with cell therapy, and for monitoring disease progression in a subject with cancer administered a cellular therapy and a therapeutic agent - Patent application The present invention provides a method for predicting the responsiveness of a subject with cancer to treatment with cell therapy. In some embodiments, the cell therapy is a CAR-T or neoantigen specific An exemplary cell therapy is described, for example, in June et al. (2018) Science 359 , 1361-1365 and Guedan et al. (2019) Annu. Rev. Immunol. 37:145-171 In some embodiments, the methods include administering a labeled CD8 binding agent described herein. and binding the labeled CD8-binding agent to CD8 in the tumor tissue of the subject. + Binding to T cells detecting binding, wherein detecting binding indicates that the subject is likely to respond to the cell therapy. In some embodiments, the method comprises administering a labeled CD8 binding agent described herein. and administering the labeled CD8 binding agent to the subject's tumor tissue. + with T cells detecting binding, wherein detecting binding indicates that the subject is in need of treatment with the cell therapy. In some embodiments, the cell therapy comprises one or more of the compounds described herein. It is administered in combination with immunotherapeutic and / or chemotherapeutic agents.

[0198] Also provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering to a subject a CD8 binding agent described herein. and administering the labeled CD8 binding agent to the tumor tissue of interest at a first time point and a second time point. Woven CD8 + In some embodiments, the method comprises detecting binding to a T cell. The method further comprises administering a therapeutically effective amount of a cell therapy.

[0199] As used herein, a patient with cancer who has previously been or is currently undergoing treatment with cell therapy is referred to as a "cell therapy patient." In some embodiments, methods are provided for monitoring the therapeutic progress of a subject. The therapy is CAR-T or neo-antigen-specific T cell therapy. (a) administering a labeled CD8 binding agent to a subject prior to administering a cell therapy, CD8-binding agents and tumor tissue CD8 + (b) detecting binding to T cells; (c) administering the labeled CD8 binding agent to the subject at a time point after administration of the cell therapy. The labeled CD8-binding agent and the CD8 in the tumor tissue were then administered to the + A steroid that detects binding to T cells and (d) CD8 in tumor tissue before and after administration of cell therapy. + T cell labeling In some embodiments, the method includes determining the mechanism of treatment failure, e.g., , tumor CD8 + This may be due to cell loss, exhaustion, and / or loss of therapeutic efficacy. It is used to explain how

[0200] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 1 24 I, 18 F, 68 The tumor tissue is labeled with a CD8-binding agent. Woven CD8 + Binding to T cells is detected by PET or PET / CT. In embodiments, the CD8 binding agent is 18 Anti-CD8 VH conjugated to F-label H. In some embodiments, the CD8 binding agent is administered via a compound of formula (I): 1 8 F]-aluminum fluoride complex conjugated to anti-CD8 VHH. In one embodiment, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, the sequence CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR comprising the amino acid sequence of SEQ ID NO: 11 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 6. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. Includes.

[0201] In some embodiments, the CD8 binding agent is administered to a subject in a repeated predictive or monitoring fashion. In some embodiments, the dose is any value between these values. At least approximately 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, including any value or range The method is repeated or the subject is Be monitored.

[0202] Methods for treating autoimmune diseases or conditions, transplant rejection, and graft-versus-host disease The CD8 binding agents described herein have high sensitivity and low immunogenicity, allowing for the prevention of disease progression. to monitor, predict response to immunotherapy, and / or autoimmune Monitoring the treatment progress of subjects with a disease or condition, transplant rejection, or graft-versus-host disease In some embodiments, the immunotherapy is an immunosuppressant.

[0203] As used herein, an autoimmune disease or condition (e.g., autoimmune arthritis, colitis, parsley, Treatment and disease progression in subjects with ACK disease, transplant rejection, or graft-versus-host disease All such diseases involve damaging inflammatory processes. As part of the CD8 + T cells. Petrelli & Femke, CD8 + T cells in human auto immune arthritis: the usual suspects; Nature Reviews Thumatology 12:421-428 (201 6). Such methods involve the use of labeled CD8-binding antibodies with or without intervention. administering a therapeutically active agent to a subject, and measuring labeled C at a first time point and a second time point. D8-binding agents and tissue CD8 +Some methods include detecting binding to T cells. In an embodiment, CD8 from the first time point and the second time point + Increased T cells may contribute to autoimmune diseases or condition, transplant rejection, or graft-versus-host disease has progressed. In this context, interventions to treat autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease are The treatment is administered prior to the labeled CD8 binding agent, and the first time point is an autoimmune disease or following administration of interventional therapy to treat the condition, transplant rejection, or graft-versus-host disease, or The first time point is after administration of a labeled CD8 binding agent, and the second time point is after the first time point. In embodiments, the CD8 + T cell levels compared to the first time point A lower value indicates positive therapeutic progress (e.g., beneficial or desired clinical outcome). In some embodiments, the CD8 + T cell levels at the first and second time points A higher relative risk is associated with a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical results). In some embodiments, tissue CD8 + T cell levels are third, fourth, or In some embodiments, tissue CD8 at the fifth or fifth subsequent time point is detected. + Lower levels of T cells compared to the first time point may indicate a lack of treatment progression (e.g., In some embodiments, the disease at a later time point is CD8 in affected tissue + Higher levels of T cells compared to the first time point indicate treatment progression. In some embodiments, the method are used to describe the mechanism of treatment failure. In some embodiments, the time points are at least Approximately 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months , 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years The intervals are longer than usual.

[0204] Also used herein are autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease. Methods for predicting the responsiveness of a subject to immunotherapeutic agents (e.g., immunosuppressants) In some embodiments, the method comprises administering to a subject a labeled CD8 binding agent described herein. and administering the labeled CD8 binding agent to the diseased tissue of the subject. + T cells detecting binding to the immunotherapeutic agent, wherein detecting binding indicates the likelihood that the subject will respond to the immunotherapeutic agent. In some embodiments, the methods include using a labeled CD8 binding antibody described herein. and administering a labeled CD8 binding agent to the diseased tissue of the subject. + detecting binding to T cells, wherein detecting binding indicates that the subject is In some embodiments, the method further comprises administering to the subject in whom binding is detected: The method further comprises administering a therapeutically effective amount of an immunotherapeutic agent.

[0205] As used herein, an autoimmune disease or condition that has received or is receiving an immunotherapeutic agent, Further disclosed are methods for monitoring the treatment progress of a subject with transplant rejection or graft-versus-host disease. In some embodiments, the method further comprises (a) administering a labeled immunotherapeutic agent to the subject prior to administering the immunotherapeutic agent. A CD8-binding agent is administered to a subject, and the labeled CD8-binding agent and CD8 in the diseased tissue are detected. + T thin (b) detecting binding to the cells; (c) administering an immunotherapeutic agent; At a time point after administration of the agent, a labeled CD8 binding agent is administered to the subject, and the labeled CD8 binding agent and CD8 in diseased tissue + detecting binding to T cells; and (d) administering an immunotherapeutic agent. CD8 in pre- and post-tumor tissues + measuring differences in labeling of T cells. In embodiments, CD8 in diseased tissue at time points after administration of the immunotherapeutic agent. + T cell levels are A lower level compared to the time point before administration of the immunotherapy agent indicates positive treatment progress (e.g., beneficial effect). or a desired clinical outcome). In some embodiments, the disease progression at time points after administration of the immunotherapeutic agent is CD8 in affected tissue + Higher levels of T cells compared to before administration of immunotherapy indicates a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical results). Morphologically, tissue CD8 + T cell levels may be 1, 2, 3, 4 or more times In some embodiments, the method provides a method for determining the mechanism of treatment failure. In some embodiments, the time point is at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, Spaced apart by 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or longer than 3 years.

[0206] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 1 24 I, 18 F, 68 The CD8-binding agent is labeled with Ga, and the tumor tissue is then bound to the labeled CD8-binding agent. CD8 + Binding to T cells is detected by PET or PET / CT. In one embodiment, the CD8 binding agent is 18 Anti-CD8 VHH conjugated to F-label In some embodiments, the CD8 binding agent is linked via a compound of formula (I) 18 F]-aluminum fluoride complex. In embodiments, the anti-CD8 VHH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a C DR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 12 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO:4. include.

[0207] In some embodiments, the method comprises treating kidney transplant rejection, liver transplant rejection, heart transplant rejection, or cardiac transplant rejection. In some embodiments, the method is used in the treatment of transplant rejection, such as lung transplant rejection. Autoimmune diseases such as SLE, vasculitis, and demyelinating neuritis, including multiple sclerosis Used for diseases or conditions.

[0208] In some embodiments, the immunotherapeutic agent is an immunosuppressant. Suitable immunosuppressants include: These include, but are not limited to, prednisone, cyclophosphamide, cyclosporine, and micoflurane. benzocaine mofetil, ibrutinib, ruxolitinib, and TNF Alpha antibodies, e.g., adalimumab, etanercept, golimumab, and infliximab Examples include biologics such as ximab.

[0209] In some embodiments, the CD8 binding agent is administered to a subject in a repeated predictive or monitoring fashion. In some embodiments, the dose is any value between these values. At least approximately 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, including any value or range The method is repeated or the subject is Be monitored.

[0210] In some embodiments, the CD8 binding agent is a therapeutic agent for treating cancer, an autoimmune disease or condition, transplantation, or the like. Allows for serial evaluation of lymphoid tissues and organs involved in rejection or graft-versus-host disease In some embodiments, the level detected from the CD8 binding agent in the subject can be The signal or signal can be correlated with other imaging techniques (e.g., MRI). In some embodiments, the level or signal detected from the CD8 binding agent in the subject nals with blood and / or tissue biomarkers (e.g., tissue biopsy biomarkers) It can be correlated.

[0211] In some embodiments, the CD8 binding agent is a CD8 antibody, e.g., a PET, SPECT, or SIRT. This allows for multiplexed imaging with other imaging scans, such as enthigraphy scans.

[0212] In some embodiments, the imaging data obtained using the CD8 binding agent is analyzed using MRI. , correlate with data from other radiological methods such as CT, ultrasound, or x-ray.

[0213] Pharmaceutical Composition Also, CD8 binding agents such as anti-CD8 antibodies (e.g., anti-CD8 VHHs), or Sequences encoding CD8 binding agents, such as anti-CD8 antibodies (e.g., anti-CD8 VHHs) In some embodiments, a composition is provided comprising a pharmaceutical formulation comprising a polynucleotide comprising: The composition may comprise one or more CD8 binding agents that bind to CD8 or one or more CD8 binding agents that bind to the CD8-binding domain. Such compositions may contain a number of polynucleotides, including buffers, as are well known in the art. The composition may further comprise a suitable carrier such as a pharmaceutically acceptable excipient.

[0214] In some embodiments, the CD8 binding agents described herein (e.g., labeled CD8 binding agents) and a pharmaceutically acceptable carrier. In some embodiments, any one of the labeled CD8 binding agents described herein, such as and one or more anti-inflammatory drugs such as methionine and / or N-acetyltryptophan. Pharmaceutical formulations are provided that include an oxidized compound. In some embodiments, the pharmaceutical formulation includes a histidine , methionine, N-acetyltryptophan, and / or sucrose. In embodiments, the pharmaceutical formulation comprises histidine, methionine, N-acetyltryptophan, and and sucrose.

[0215] Pharmaceutical preparations of CD8 binding agents as described herein include those having the desired purity. Such antibodies are mixed with one or more optional pharmaceutically acceptable carriers. , prepared in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences ces 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally a pharmaceutically acceptable carrier that is nontoxic to recipients at the dosages and concentrations prescribed These include, but are not limited to: phosphate, citrate, and other organic acids; buffering agents such as ascorbic acid, N-acetyltryptophan, and Antioxidants containing methionine; preservatives (octadecyldimethylbenzylammonium chloride) Benzalkonium chloride; Benzethonium chloride; phenol; butyl or benzyl alcohol; methyl or propyl paraben Alkylparaben; Catechol; Resorcinol; Cyclohexanol; 3-Pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum alcohols Proteins such as phospholipids, ... Which hydrophilic polymers; glycine, glutamine, asparagine, histidine, arginine, or amino acids such as lysine; monosaccharides, disaccharides, and glucose , mannose, or other carbohydrates including dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; sodium salt-forming counterions such as; metal complexes (e.g., Zn-protein complexes); and / or is a non-ionic surfactant such as polyethylene glycol (PEG). Pharmaceutically acceptable carriers include soluble neutral active hyaluronidase glycoprotein (sH ASEGP), e.g., rHuPH20 (HYLENEX®, Baxter Human soluble PH-20 hyaluronider, such as PH-20 hyaluronidase (HHA) or ... Further examples include interstitial drug dispersion agents such as glycoproteins. Certain exemplary sHASEGPs and methods of use are described in U.S. Patent Application Publication No. 2005 / 022999. 60186 and 2006 / 0104968. In such cases, sHASEGP is coupled to one or more additional glycosaminoglycans, such as chondroitinase. Combine with aminoglycanase.

[0216] Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and International Publication No. 2002 / 002264. The latter formulation is described in the pamphlet of 006 / 044908. Contains histidine-acetate buffer.

[0217] The formulations herein may also be used, if desired, to treat the particular indication being treated (e.g., cancer, autoimmune disease, or other conditions). more than one for an autoimmune disease or condition, transplant rejection, or graft-versus-host disease Active ingredients (e.g., immunotherapeutic agents), preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide a statin Such active ingredients are preferably combined in amounts effective for the purpose intended. It exists.

[0218] The active ingredient may be prepared, for example, by coacervation techniques or by interfacial polymerization. Microcapsules, e.g., hydroxymethylcellulose or gelatin-microcapsules, respectively Microcapsules and poly(methylmethacylate) microcapsules capsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres), , microemulsions, nanoparticles, and nanocapsules), or macroemulsions Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0219] Sustained release formulations may also be prepared. Suitable examples of sustained release formulations include those containing antibodies. Semipermeable matrices of solid hydrophobic polymers are included, which matrices can be formed into shaped articles, e.g. For example, in the form of a film or microcapsules.

[0220] Formulations to be used for in vivo administration are generally sterile. Sterility can be achieved, for example, by sterile filtration. This can be easily achieved by filtration through a membrane.

[0221] Manufactured Products and Kits In some embodiments, the present invention is directed to treating a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) to immunotherapeutic agents, Disease (e.g., cancer, autoimmune disease or condition, transplant rejection, or graft-versus-host disease) to monitor disease progression in subjects with, and / or to treat, a disease (e.g., cancer, autoimmune disease, progress in the treatment of subjects with a comorbid condition (e.g., an immune disease or condition, transplant rejection, or graft-versus-host disease) An article of manufacture or kit containing materials useful for monitoring the line is provided.

[0222] In some embodiments, the article of manufacture or kit comprises a CD8 binding agent or In some embodiments, the article of manufacture or The kits may contain one or more of the CD8 binding agents or compositions described herein. In some embodiments, the kit comprises a container containing the nucleic acid to be loaded. The present invention also includes cells of a cell line that produces a CD8-binding agent (e.g., an anti-CD8 antibody).

[0223] In some embodiments, the kit or article of manufacture comprises an anti-CD8 VHH. In some embodiments, the kit or article of manufacture may include a labeled CD8 binding agent, e.g., a detectable label. In some embodiments, the kit comprises an immunoconjugate comprising an anti-CD8 antibody (e.g., , anti-CD8 VHH) and a labeled CD8 binding agent. The kit or article of manufacture comprises a chelating agent of formula (I) and 18 F]-aluminum fluoride The present invention further includes reagents for preparing labeled CD8 binding agents, such as fluorophore complexes.

[0224] In some embodiments, the labeled CD8 binding agent is 18 Conjugated to F label In some embodiments, the CD8 binding agent is a compound of formula (I): Through things 18 Anti-CD8 VH conjugated to [F]-aluminum fluoride complex H. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 7. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 1 In some embodiments, the anti-CD8 VHH comprises a CDR3 comprising the amino acid sequence In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4. It contains the amino acid sequence of

[0225] In some embodiments, the kit includes one or more positive controls, such as CD8 (or or its fragments) or CD8 + In some embodiments, the kit includes a negative control, e.g., For example, it includes a surface or solution that is substantially free of CD8.

[0226] In some embodiments, the article of manufacture or kit comprises a container and a Suitable containers include, for example, bottles, vials, syringes, and the like. Examples include syringes, IV infusion bags, etc. Containers can be made of various materials such as glass or plastic. The container itself may be formed from a variety of materials for the treatment, prevention, and / or prevention of cancer. or a composition in combination with another composition effective for diagnosis, and may have a sterile access port (e.g., the container may be connected to an intravenous fluid bag, or a subcutaneous injection (It may be a vial with a stopper that can be pierced with a needle). At least one agent is a CD8 binding agent described herein. The accompanying document describes a method for predicting the responsiveness of a subject with cancer to an immunotherapeutic agent. , for monitoring disease progression in subjects with cancer, and / or for use in subjects with cancer The indication is that the drug is used to monitor the progress of treatment.

[0227] The article of manufacture or kit may further comprise: (a) a first container having a composition contained therein; wherein the composition comprises a first container comprising a CD8 binding agent described herein; and (b a second container having the composition contained therein, the composition containing a further cytotoxic agent or In some embodiments, the composition may include a second container that contains a therapeutic agent or otherwise. The therapeutic agent is an immunotherapeutic agent as described herein.

[0228] The articles of manufacture or kits provided herein may be used to treat diseases (e.g., cancer, autoimmune diseases, etc.). or conditions, transplant rejection, or graft-versus-host disease) to immunotherapeutic agents To predict responsiveness, the disease (e.g., cancer, autoimmune disease or condition, transplant rejection, or graft-versus-host disease), and / or to monitor disease progression in subjects with is a disease (e.g., cancer, autoimmune disease or condition, transplant rejection, or graft-versus-host disease) The compositions can be used to monitor the progress of treatment of a subject with Additionally, the article of manufacture may contain Bacteriostatic Water for Injection (BWFI ), phosphate-buffered saline, Ringer's solution, and dextrose solution, among other pharmaceutically acceptable The article of manufacture may further comprise a second (or third) container containing a buffer solution to be used. Commercial and user-friendly, including other buffers, diluents, filters, needles, and syringes. It may further comprise other materials as desired from the viewpoint.

[0229] Illustrative Embodiments The present application provides the following embodiments. 1. A CD8-binding agent containing a variable domain of the heavy chain of a heavy chain antibody (VHH domain). and a K of about 1 nM or lower D CD8 binding specifically binds to human CD8 Sexually active agent. 2. k of approximately 0.002 / sec or lower off Binds to human CD8 in the embodiment A CD8 binding agent according to embodiment 1. 3. KD and / or k off single-arm human CD8α / human CD8β-Fc fusion a conjugated protein (e.g., human CD8α and CD8β fused to one polypeptide chain of Fc) and a single-chain polypeptide containing human CD8β) as a reagent for surface plasmon resonance analysis. The CD8 binding agent of embodiment 1 or 2, wherein the CD8 binding agent is determined by: 4. K of approximately 1 nM or lower D 1, which binds to cynomolgus monkey CD8 at 4. The CD8 binding agent according to any one of claims 1 to 3. 5. k of approximately 0.004 / sec or lower off binds to cynomolgus monkey CD8 10. The CD8 binding agent according to any one of embodiments 1 to 4. 6. K D and / or k off Single-arm cynomolgus CD8α / cynomolgus CD8β-Fc fusion proteins (e.g., CD8β-Fc fusion proteins fused to one polypeptide chain of Fc) , a single-chain polypeptide containing cynomolgus monkey CD8α and cynomolgus monkey CD8β) as a reagent. 6. The CD of embodiment 4 or 5, as determined by surface plasmon resonance using 8-binding agent. 7. CD8 + Any one of embodiments 1 to 6, which neither stimulates nor inhibits T cell activation 2. The CD8 binding agent according to claim 1. 8. CD8 + 8. A CD according to any one of embodiments 1 to 7, which does not induce T cell proliferation. 8-binding agent. 9. CD4 + The CD8 binding antibody of any one of embodiments 1 to 8, which does not bind to T cells. Synergistic agent. 10. The method of any one of embodiments 1 to 9, wherein the VHH domain is a llama VHH. CD8 binding agents. 11. Any one of embodiments 1 to 10, wherein the VHH domain is humanized. CD8 binding agent. 12. The VHH domain contains Arg25, Lys42, Gln44, Val45, and Le u46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Human CD8 containing Arg93, Leu94, Gly95, Asp96, and Thr97 11. Specific binding to the alpha epitope, with amino acid numbering according to SEQ ID NO: 13. 10. The CD8 binding agent according to any one of the preceding claims. 13. The VHH domain has a complementarity determining region comprising the amino acid sequence of SEQ ID NO: 6 or 7. (CDR)1; CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and SEQ ID NO: 10 13. The CD45 of embodiment 12, comprising a CDR3 comprising any one of the amino acid sequences of to 12. 8-binding agent. 14. The VHH domain is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and CDR2 comprising the amino acid sequence of SEQ ID NO: 8 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and CDR2 comprising the amino acid sequence of SEQ ID NO: 9 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, and CDR2 comprising the amino acid sequence of SEQ ID NO: 9 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and CDR2 comprising the amino acid sequence of SEQ ID NO: 9 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 14. The CD8 binding agent of embodiment 13, comprising: 15. The VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR3 comprising the amino acid sequence of SEQ ID NO: 13. 14. The CD8 binding agent of embodiment 13. 16. The embodiment wherein the VHH domain comprises L49A, and the numbering is according to the Kabat numbering system 16. The CD8 binding agent according to any one of 1 to 15. 17. The VHH domain contains a V89T substitution, a T110Q substitution, an S112Q substitution, and A114 addition, and 17. The CD8 binding agent according to any one of embodiments 1 to 16, according to the abat numbering. 18. A VHH domain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. A CD8 binding agent according to any one of embodiments 1 to 17. 19. The CD8-binding antibody of any one of embodiments 1 to 18, which does not contain an Fc region. Agent. 20. A single molecule encoding a CD8 binding agent of any one of embodiments 1 to 19. Isolated nucleic acids. 21. An expression vector comprising the nucleic acid of embodiment 20. 22. A host cell comprising the nucleic acid of embodiment 20 or the expression vector of embodiment 21. Principal cell. 23. The host cell of embodiment 22, which is a eukaryotic cell. 24. The host cell of embodiment 23, which is a mammalian cell. 25. The host cell of embodiment 24, which is an Expi293 cell. 26. The host cell of embodiment 22, which is a prokaryotic cell. 27. A method for making a CD8 binding agent, comprising: a) Inducing a host cell according to any one of embodiments 22 to 26 under conditions in which the agent is produced. culturing the culture under conditions; and b) recovering the CD8 binding agent produced by the host cell A method comprising: 28. Any of embodiments 1 to 19, wherein the VHH domain is conjugated to a label. The CD8 binding agent according to any one of the above. 29. The method of claim 28, wherein the label is a fluorescent dye, a radionuclide, or an enzyme. D8 binding agents. 30. The CD8 binding agent of embodiment 29, wherein the label is a radionuclide. 31. A radionuclide is 18 F, 89 Zr, 99m Tc, 67 Ga, 68 Ga, 64 C u, 52 Mn, 111 In, or 124 31. The CD8-binding antibody of embodiment 30, wherein the antibody is I. Agent. 32. The VHH domain is conjugated to a label via a chelating moiety. The CD8 binding agent of any one of embodiments 28 to 31. 33. A chelating moiety is covalently linked to a VHH domain via a lysine residue. 33. The CD8 binding agent of embodiment 32, wherein 34. The label forms a complex with a metal, and the complex is chelated by a chelating moiety. 34. The CD8 binding agent of embodiment 32 or 33, 35. Signs 18 35. The CD of embodiment 34, wherein F is F and the metal is aluminum. 8-binding agent. 36. The chelating moiety has the formula (I):

[0230] [ka] 36. The CD8 binding agent of embodiment 35, which is a compound of the formula: 37. Subject CD8 +1. A method for detecting a cell, comprising: a) administering a labeled CD8 binding agent according to any one of embodiments 28 to 36 to a subject; providing the b) Labeled CD8 binding agent and CD8 of the subject + Detecting binding to cells and detection of binding is by CD8 + indicating the presence of cells, method. 38. A labeled CD8 binding agent and a subject's CD8 + detecting binding to cells , subject's CD8 + 38. The method of embodiment 37, comprising imaging the cells. 39. Subject CD8 + Cellular imaging is performed on the subject using positron emission tomography (PET) scan or positron emission tomography / computed tomography (PET / CT) scan 39. The method of embodiment 38, comprising performing 40. CD8 + The cells are CD8 + Any one of embodiments 37-39, wherein the T cell The method described below. 41. CD8 + The cells are CD8 + Any one of embodiments 37 to 40, wherein the cell is a tumor cell. The method described in the first paragraph. 42. The detecting step is performed within about one day or less after administration. The method according to any one of embodiments 37 to 41, wherein 43. The method of any one of embodiments 37 to 42, repeated one or more times. method. 44. Embodiment 43, wherein the administration is repeated about 1 day after the previous administration of the CD8 binding agent. The method described below. 45. The method of embodiment 43 or 44, repeated 1 to 4 times per year. 46. ​​Any one of embodiments 43 to 45, repeated for a period of more than one year. The method described in the first paragraph. 47. Any one of embodiments 37 to 46, having a sensitivity of about 1 nM to about 30 nM. The method described. 48. Any one of embodiments 37-47, wherein the subject is a human or non-human primate. The method described below. 49. As described in embodiment 48, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey. How to do it. 50. The method of embodiment 48, wherein the subject is a human. 51. The method of any one of embodiments 37 to 50, wherein the subject has cancer. 52. The embodiment in which the subject has an autoimmune disease, transplant rejection, or graft-versus-host disease 37 to 50. A method according to any one of claims 37 to 50. 53. Response of subjects with cancer to immunotherapy, cell therapy, or cancer vaccines 1. A method for predicting response to a test, comprising: a) administering a labeled CD8 binding agent according to any one of embodiments 28 to 36 to a subject; providing the b) A labeled CD8 binding agent and a CD8 binding agent in the tumor tissue of interest + Detecting binding to T cells Steps to take detecting binding, wherein the subject is likely to respond to the immunotherapeutic agent, cell therapy, or cancer vaccine. indicates high potential, method. 54. (c) administering a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or Administering a cancer vaccine 54. The method of embodiment 53, further comprising: 55. A method for monitoring disease progression in a subject having cancer, comprising: a) administering a labeled CD8 binding agent according to any one of embodiments 28 to 36 to a subject; providing the b) combining the labeled CD8 binding agent with the tumor tissue of the subject at a first time point and a second time point. CD8 + Detecting binding to T cells A method comprising: 56. (c) Administering a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or cancer vaccine to a subject. and measuring CD8 in the tumor tissue at the second time point. + T cell levels CD8 in tumor tissue at the first time point + higher than the level of T cells, The method of embodiment 55. 57. Have received or are currently receiving immunotherapy, cell therapy, or cancer vaccine 1. A method for monitoring the treatment progress of a subject having cancer, comprising: i) any of embodiments 28 to 36 in conjunction with an immunotherapeutic agent, a cell therapy, or a cancer vaccine; administering to a subject a labeled CD8 binding agent according to any one of the preceding claims; and ii) measuring the CD8 binding activity of the labeled CD8 binding agent with the CD8 binding activity of the tumor tissue at the first and second time points; 8 + Detecting binding to T cells A method comprising: 58. The labeled CD8 binding agent is used as a precursor to an immunotherapy, cell therapy, or cancer vaccine. the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, cell therapy agent, or or cancer vaccine, and the second time point is before administration of an immunotherapy, cell therapy, or 58. The method of embodiment 57, wherein the method is after administration of a cancer vaccine. 59. Immunotherapeutic agents, cell therapies, or cancer vaccines are administered prior to the labeled CD8-binding agent. the first time point being after administration of the immunotherapy, cell therapy, or cancer vaccine. and after administration of a labeled CD8 binding agent, wherein the second time point is after the first time point. 58. The method of embodiment 57. 60. Any of embodiments 53-54 and 56-59, wherein an immunotherapeutic agent is administered to the subject. The method according to any one of the preceding claims. 61. The immunotherapeutic agent is an anti-PDL1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, or a TIGI T antagonist, anti-CSF-1R antibody, anti-CSF-1R antagonist, anti-CEA antibody , anti-CEA antagonist, anti-CTLA4 antibody, CTLA4 antagonist, anti-OX40 61. The method of embodiment 60, wherein the antibody is an antibody or an OX40 agonist. 62. The method of embodiment 61, wherein the immunotherapeutic agent is an anti-PD-L1 antibody. 63. The method of embodiment 62, wherein the anti-PD-L1 antibody is atezolizumab. 64. Clinical trials in which anti-PD-L1 antibodies are administered in combination with one or more therapeutic agents 64. The method of embodiment 62 or 63. 65. One or more therapeutic agents include TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obine (tuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COT ELLIC® (cobimetinib), ALECENSA® (alectinib) ), KADCYLA® (ado-trastuzumab emtansine), HERCEP TIN® (trastuzumab), PERJETA® (pertuzumab) , polatuzumab, IFN-alpha, anti-CD40 agent, anti-OX40 antibody, OX40 agonist anti-CSF-1R antibody, anti-CEA antibody, IDO inhibitor, or anti-TIGIT antibody 65. The method of embodiment 64. 66. The method of embodiment 60, wherein the immunotherapeutic agent is a cytokine. 67. The cytokine is IL2, genetically modified IL2, IL15, or genetically modified IL 67. The method of embodiment 66, wherein the compound is L15. 68. The immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD3. The method of embodiment 60. 69. The immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD16. 61. The method of embodiment 60. 70. An embodiment in which the bispecific antigen-binding molecule is an antibody or an antigen-binding fragment thereof. 69. The method according to claim 68 or 69. 71. Embodiment 69, in which the bispecific antigen-binding molecule specifically binds to CD16A. Or the method described in 70. 72. The method of embodiment 60, wherein the immunotherapeutic agent is a dendritic cell modulator. 73. The embodiment in which the immunotherapeutic agent is a dendritic cell activator or a dendritic cell growth factor. 72. The method according to claim 72. 74. The method of embodiments 53-54 and 56-59, wherein a cancer vaccine is administered to the subject. The method according to any one of the preceding claims. 75. As described in embodiment 74, wherein the cancer vaccine is a personalized cancer vaccine (PCV). How to do it. 76. Any of embodiments 53-54 and 56-59, in which a cell therapy is administered to a subject. The method according to any one of the following: 77. The method of embodiment 76, wherein the cell therapy is CAR-T or neoantigen-specific T cells. The method described. 78. Immunotherapeutic agents for subjects with autoimmune diseases, transplant rejection, or graft-versus-host disease 1. A method for predicting responsiveness to a drug, comprising: a) administering a labeled CD8 binding agent according to any one of embodiments 28 to 36 to a subject; providing the b) A labeled CD8 binding agent and a CD8 binding agent in a diseased tissue of interest. + Detecting binding to T cells Steps to take wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. method. 79. (c) administering a therapeutically effective amount of an immunotherapeutic agent to a subject in whom binding is detected. 79. The method of embodiment 78, further comprising: 80. Monitor disease progression in subjects with autoimmune disease, transplant rejection, or graft-versus-host disease. 1. A method for monitoring a signal, comprising: a) administering a labeled CD8 binding agent according to any one of embodiments 28 to 36 to a subject; providing the b) combining a labeled CD8 binding agent with a diseased tissue of the subject at a first time point and a second time point; CD8 + Detecting binding to T cells and CD8 from the first time point and the second time point. + Increased T cells are a key factor in preventing autoimmune diseases, indicates the progression of transplant rejection or graft-versus-host disease, method. 81. (c) administering a therapeutically effective amount of an immunotherapeutic agent to the subject. and measuring CD8 in the diseased tissue at a second time point. + T cell levels at the first time point CD8 in diseased tissue + Lower than T cell levels The method of embodiment 80. 82. Have received or are receiving immunotherapy, have an autoimmune disease, transplant rejection, or 1. A method for monitoring the treatment progress of a subject with graft-versus-host disease, comprising: i) a labeled CD8 binding agent according to any one of embodiments 28 to 36 in combination with an immunotherapeutic agent; administering the combined agent to a subject; and ii) measuring the CD8 binding agent and the CD8 binding activity of the diseased tissue at the first and second time points; 8 + Detecting binding to T cells A method comprising: 83. A labeled CD8 binding agent is administered before the immunotherapeutic agent, and the first time point is the second time point is after administration of the CD8 binding agent but before administration of the immunotherapeutic agent; 83. The method of embodiment 82, wherein the administration of 84. The immunotherapeutic agent is administered before the labeled CD8 binding agent, and the first time point is the first time point is after administration of the therapeutic agent and after administration of the labeled CD8 binding agent; 83. The method of embodiment 82, which is after time point 1. 85. A labeled CD8 binding agent and a subject's CD8 + Detecting binding to T cells is the target CD8 + Any one of embodiments 53-84, comprising imaging T cells. The method described below. 86. Subject CD8 + T cell imaging is performed on subjects using positron emission tomography (PET) ) scan or positron emission tomography / computed tomography (PET / CT) scan 86. The method of embodiment 85, comprising performing: 87. Another imaging session was performed within approximately 48 hours of the first imaging session using a labeled CD8-binding agent. Performing a scan (e.g., PET, SPECT, or scintigraphy scan) 87. The method of any one of embodiments 53 to 86, further comprising the step of: 88. The method of embodiments 55-77 and 80-, wherein the subject is monitored for at least one year. 87. The method according to any one of claims 87 to 87. 89. Identifying gut microbial strains associated with response to immunotherapy treatment 1. A method for a) obtaining gut microbiome samples from a population of subjects with cancer; Thus, the population includes subjects who are responsive to treatment with immunotherapeutic agents and subjects who are not responsive to treatment with immunotherapeutic agents. Steps, including non-responsive objects; b) Gut microbiome samples from subjects who are responsive to treatment and subjects who are not responsive to treatment Analyzing a gut microbiome sample from the subject; and c) identifying gut microbial strains associated with subjects who are responsive to the treatment and the responsiveness is determined by the use of a labeled CD8 binding agent according to any one of embodiments 28 to 36. and CD8 in the tumor tissue of interest. + It is determined by detecting binding to T cells. indicates that the subject is responsive to the immunotherapeutic agent. method. 90. Microbial strains, including those in the gut that are associated with responsiveness to immunotherapeutic agents. 90. The method of embodiment 89, further comprising preparing an iome-based drug. 91. The method of embodiment 89 or 90, wherein the immunotherapeutic agent is an anti-PD-1 antibody. . 92. The method of embodiment 89 or 90, wherein the immunotherapeutic agent is an anti-PD-L1 antibody. Law. 93. The method of embodiment 92, wherein the anti-PD-L1 antibody is atezolizumab. 94. A kit comprising a labeled CD8 binding agent according to any one of embodiments 28 to 36. tt. 95. A method for preparing a labeled CD8 binding agent, comprising: Conjugated to the VHH domain of the CD8 binding agent according to any one of embodiments 1 to 19. gating to provide a conjugate; and 18 Contains F and contacting the labeled CD8 binding agent with an aluminum fluoride complex containing the compound. wherein the chelating moiety has the formula (I):

[0231] [ka] The method is a compound of 96. The conjugate is reacted with aluminum fluoride in the presence of one or more antioxidant compounds. 96. The method of embodiment 95, wherein the compound is contacted with a tetrahydrofuran complex. 97. The one or more antioxidant compounds may be methionine and / or N-acetyl- 97. The method of embodiment 96, comprising tryptophan. 98. A CD8 binding agent according to any one of embodiments 1 to 19 and 28 to 36. and one or more antioxidant compounds. 99. The one or more antioxidant compounds may be methionine and / or N-acetylglucosamine. The pharmaceutical formulation of embodiment 98, comprising tryptophan. 100. The method of embodiment 98 or 99, further comprising histidine and sucrose. Pharmaceutical preparations of. [Example]

[0232] [Example 1] Development and characterization of VHHs against human CD8 Discovery and initial screening of anti-CD8 VHHs Llamas were incubated with two antigens: C-terminal hFc-tagged CD8α receptor (CD8α-Fc) or C A C-terminal histidine-tagged single-chain protein in which CD8α is fused to CD8β via a linker The mice were immunized with either CD8αβ-His or CD8αβ-His. A standard immunization protocol was performed as described in U.S. Pat. No. 6,015,695. Standard RT-PCR techniques were used to amplify the VHH heavy chain repertoire, The phagemid vector was cloned to construct an immune phage library. In this study, either CD8αβ-His or CD8α-Fc was injected at various concentrations, washing times, and Several rounds of in vitro selection with a phage library were performed using the same incubation and elution conditions. After three and four rounds of selection, individual phage clones were selected using E. coli. They were characterized by LISA and subjected to Sanger sequencing.

[0233] In addition, human (huCD8a-Fc) CD8 and cynomolgus monkey (cynoCD8a- The binding affinity of selected VHH antibodies to both Fc and CD8 was determined by SPR. In particular, 2C8.1-H (also referred to herein as "wt2C8") was found to be a huCD It showed acceptable affinity and binding to 8+ HPBALL cells (Figure 3).

[0234] Expression and purification of VHHs The unique sequences identified from phage panning were then cloned into mammalian gene products containing a C-terminal His tag. The expressed VHH was purified in two steps: NiSepharose and HCl. ose excel histidine-tagged protein purification resin (GE Healthcare) e), followed by size exclusion chromatography (SEC). Untagged VHHs were purified by ion exchange (SP column) or recombinant protein. Protein A resin (GORE) followed by SEC.

[0235] SPR characterization Human (single-arm, single-chain huCD8α / huCD8β-Fc) and cynomolgus monkey (single-arm, single-chain huCD8α / huCD8β-Fc) The VHH mutants were tested against both the single-chain cynoCD8α / cynoCD8β-Fc. The binding affinity was determined by surface plasmon resonance (SPR). SPR experiments were performed at 37°C. Biac using HBS-P+ (GE Healthcare) running buffer. The assay was performed using a GE Healthcare T200. Using a capture kit (GE Healthcare), 1.5 μg / mL of CD8αβ- Fc was captured and monomeric VHH was added as analyte in solution at a flow rate of 100 μL / min. VHHs were titrated using a dilution series from 100 to 0 nM. Sensorgrams were plotted in 1:1 runs. Fitting to the Muir model, K D , k on , and k off Dynamic parameters including The meter was identified.

[0236] CD8 + Identification of cell-specific VHH variants Recombinant VHHs fused to a C-terminal huIgG1 Fc were expressed, purified, and subjected to FACS. Therefore, we developed huCD8+ HPBALL cells (DS), a CD8-expressing human T-cell leukemia cell line. The cells were screened against human embryonic kidney (HE) cells obtained from ATCC (MZ, Germany). K) was used as a non-CD8-expressing control cell line. Approximately 300,000 cells were cultured at 10 % v / v fetal bovine serum and 1% v / v penicillin-streptomycin (Thermo Presence of 100 μL RPMI medium supplemented with 100 μL of 1% ethanol (Fisher Scientific) The V fused to human IgG Fc was plated in a round-bottom 96-well plate. HH mutants were incubated with cells at a concentration of 10 μg / mL for 60 minutes at 37°C. Afterwards, unbound VHHs were washed away and Alexa Fluor® 647 goat Anti-human IgG Fc antibody (Jackson Immuno Research, Inc. ) was added to the cells at a concentration of 7.5 μg / mL for 60 minutes at 37°C. Wash twice with phosphate-buffered saline (pH 7.4) supplemented with 0.5% bovine serum albumin. , analyzed on a FACSCalibur flow cytometer (BD Biosciences). Samples were analyzed in duplicate. OKT, derived from a four-chain antibody that binds to human CD8a 8-Fc served as a positive control.

[0237] The results are shown in Figure 3. This figure shows that 2C8 VHH-Fc and OKT8-Fc They have been shown to have comparable affinity and binding to HPBALL cells.

[0238] Binding to whole blood cells and PBMCs The 2C8 VHH was used for further FACS analysis of human whole blood and corresponding PBMC samples. VHHs were expressed as His-tagged monomers and purified using ALEXA FLUOR ( Directly labeled with 647.

[0239] Briefly, whole blood samples from four healthy donors (Health Center, Genentech) Blood samples were collected at room temperature and processed within 30 minutes of collection. For each donor, 1 mL of the sample was directly A 4 mL sample was kept for staining and was then transferred to a Peripheral Blood Mononuclear Cell (PBMC) isolation (Fico) tube prior to staining. To avoid nonspecific binding, whole blood or PBMCs were used. or 10 cells per 1000 μL, respectively, before staining. 7 20 μL of FcR per unit Incubated with blocking reagent (Miltenyi Biotech) For flow cytometry staining, 100 μL of whole blood or 5 x 10 4 PBMCs The fluorescently labeled antibodies were dispensed into a 96-deep well plate according to the manufacturer's protocol (Ther According to Fisher Scientific, ALEXA FLUOR (registered trademark) Prepared by EDC-NHS-mediated conjugation with AF647 dye Made.

[0240] OKT8-AF647 or VHH-AF647 mutant (20 ng / mL), anti-CD1 4 VioBlue (Miltenyi Biotech; 1 / 25), anti-CD16 P erCP Cy5.5(Becton-Dickinson;1 / 200), anti-CD4 VioBright-FITC (Miltenyi Biotech;1 / 50), anti-C D3 APC-Vio770 (Miltenyi Biotech; 1 / 50) was used for pre- The mixed antibody cocktail was added at room temperature for 10 minutes. The sample was then added to 2 mL of red blood cells. Resuspend in globule lysis solution (Becton Dickinson), mix well, and incubate at room temperature for 10 minutes. After incubation for 1 hour, the mixture was centrifuged at 1500 rpm for 5 minutes. The tubes were washed with 1x PBS, 0.5% BSA, and then MacsQuant10 analytes were added. Cell acquisition was performed using a microscope (Miltenyi Biotech).

[0241] The same protocol was followed for PBMC staining, except that the red blood cell lysis step was omitted. was applied to.

[0242] Results using whole blood cell samples are shown in Figure 4. OKT8 and 2C8 VH H is CD8 + Strong staining of T cells and CD3 - Low levels of cells (e.g., NK cells) Similar results were obtained in experiments using PBMC samples. 2C8 VHH was expressed in whole blood (containing polynuclear and mononuclear cells) and PBMCs. (containing only mononuclear cells) + CD8 + Potentially only in T cell populations This confirms the specificity for human CD8.

[0243] Structural characterization To determine the epitope of 2C8 on CD8α, we bound it to homodimeric CD8αα. The crystal structure of 2C8 was determined (Fig. 5). 2C8 binds to the apical end of CD8α and binds to Arg2. 5, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pr o50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, These CD8α amino acid residues are in contact with Asp96 and Thr97. In the structure, each is within approximately 4.5 Å of one or more amino acid residues of 2C8. The epitope overlaps with the binding epitope of mouse CD8αβ complexed with MHCI. (Wang et al. J Immunol 2009).

[0244] Affinity maturation of VHH CDRs by NNK walk The initial affinity of 2C8 was determined by PEGylation, as described by Rashidian et al. JEM 2017. It is considered suboptimal for sensitive detection of CD8+ cells without some half-life extension mechanism. To identify mutations in 2C8 that improved affinity, we used Koen An NNK walk of the CDRs of 2C8 was performed as described in ig, et al. JBC 2015.

[0245] Briefly, each mutant contains a single mutation and the entire library is composed of VHHs. CDR NNK scan library containing all 20 amino acids at all CDR positions The library was cloned into a phagemid vector and grown at decreasing concentrations and The cells were subjected to several rounds of phage panning against CD8α-hFc with increasing wash times. VHH domains from the initial library and from the library selected in round 3 were The DNA was amplified and subjected to next-generation sequencing (NGS). NGS was performed using MiSeq (Il Three rounds were performed on the amplified DNA amplicons using a lumina instrument. Dividing the frequency of each mutation after selection by the frequency of each mutation in the initial library The concentration rate was determined by the following.

[0246] When analyzing the NGS results, the inventors found that A99G and A100fD were strongly enriched. The generation of 2C8 with the A99G and A100fD mutations was observed. , improving affinity by approximately 10-fold compared to the parent clone.

[0247] Humanization of 2C8 The present inventors have identified CDRs (30-35 (H1), 50-65 (H2), and 94-10 2(H3)) to IGHV3-23 * 2C8 was humanized by grafting it onto 04. All Vernier positions derived from the llama are also listed in their corresponding positions. In order to maintain the binding, stability, and soluble expression of VHHs, the present inventors Because of the need, several llama residues (F37, R45, G47, and L49) were added to the framework. After SPR characterization, we determined the required amount of ATP to maintain high affinity binding. It was determined that only the S71 and V78 Vernier residues are essential. The inventors found that, upon humanization, all Protein A contact sites have favorable residues. However, I noticed that it had very poor binding to Protein A resin ( (Henry et al., PLoS One 2016). We have investigated the effect of direct contact with protein A. The conformation of VHH residues important for Protein A binding, outside of the residues that bind to the VHH, is indirectly altered. The inventors investigated potential residues that could be selectively altered. The inventors identified L49 as one of the nucleotide sequences. When L49 was mutated to Ala (L49A), the promoter A significant increase in VHH recovery after purification was observed using protein A residues. (Table 3).

[0248] [Table 3]

[0249] Reduced coupling with existing ADA Previous clinical data from VHHs have shown that patients have pre-existing anti-VHH antibodies. [Cordy et al. Clin Exp Immunol 2015;Holland et al. J Clin Immunol 2013;Papa dopoulos et al. Cancer Chemother Pharmacol 2015]. Binding to existing anti-VHH antibodies was assessed, and the linkages associated with binding to these existing antibodies were identified. Four framework mutations (V89T, T110Q, S11 2Q, and A114 addition) were introduced.

[0250] To perform the VHH anti-drug antibody assay, VHH variants were added at 2 μg / mL in PBS. Maxisorp plates were coated overnight at 4°C. The plates were then resuspended in PBS + 0.5% COOH. Wash with 2% BSA + 0.1% Tween 20 (PBSBT) and incubate in 2% BSA for 2 hours at 25°C. Individual serum samples from 96 different healthy donors were diluted 1:50 and blocked with 1:10. Incubate the cells in the empty VHH-coated wells for 1-2 hours at 25°C with shaking. After washing, anti-human Fc specific HRP2 antibody (1:10,000) was added at 25°C. After washing with PBSBT, the plate was incubated with TMB substrate for 10 minutes. Color was developed for 1 h and detected at 650 nm.

[0251] As shown in Figure 6, the framework mutations were compared between the anti-CD8 VHH and the 96 human VHH. This eliminates binding to pre-existing anti-VHH antibodies pooled from healthy donors.

[0252] Overall, we found strong affinity with both human CD8α and cynomolgus monkey CD8α. Several humanized and optimized clones (v130, v142, and v 144) were developed. Figure 1 and Table 4 show the amino acid sequences of exemplary VHH clones. It has been done.

[0253] [Table 4]

[0254] Tables 5 and 6 show the αβ and 2C8v142 sequences, respectively, as determined by SPR. The affinity of 2C8v144 to human CD8α and It has high affinity to cynomolgus monkey CD8α and a relatively slow off-rate. This clone contains a W98F mutation in CDR3 compared to 2C8v144, thereby increasing the C8v142 is less susceptible to oxidation than 2C8v144, even when exposed to high levels of radiation. become.

[0255] [Table 5]

[0256] [Table 6]

[0257] Effects on T cell function CD8 + To assess the potential impact of 2C8 VHH binding on T cell function, The inventors performed an in vitro T cell proliferation assay in the presence of 2C8.v130. did.

[0258] Briefly, freshly isolated PBMCs from three healthy donors were washed with PBS 1x. and pellet 10 x 10 cells per mL. 6 The cells were resuspended in 1x PBS. Freshly prepared carboxyfluorescein succinimidyl ester (CFSE) 2.5mM Add the M working solution (Molecular Probe) and incubate at room temperature for 5 minutes. Labeling was stopped by adding 9 volumes of RPMI, 10% FBS. Then, the cells were centrifuged at 1500 rpm for 5 minutes. Washing was performed before cell counting and distribution.

[0259] 1 μg / mL anti-CD28 or 0.4 μg / mL superantigen Staphylococcus aureus lococcal) enterotoxin B (SEB) (TruCulture tubes; Myria d RBM) freshly added polyclonal stimulators with 0.2 μg / mL anti-CD3. Pre-coated round-bottom 96-well plates (Becton Dickinson) 100,000 CFSE-labeled cells were plated on a 100-well plate. To achieve this, 500,000 CFSE-labeled cells were plated in a round-bottom 96-well plate. Then, 2 μg / mL of CEF peptide pool (Mabtech) was added to each well. 10 ng / mL lipopolysaccharide ("LPS", Sigma) was administered to stimulate innate cell activity. The medium alone (RPMI, 10% FBS) was used as a negative control. Finally, for all conditions, PBS 1x, 2C8v130 VHH (final concentration 1 and 10 μg / mL) or a control Lys2 VHH (10 μg / mL ) was added in triplicate to a final volume of 200 μL per well. The Lys2 VHH binds to lysozyme and is described in De Genst, et al. JBC 2005. there was.

[0260] Additionally, to assess the potential influence of circulating human blood molecules on the efficacy of VHH binding. The FBS in the culture medium was adjusted to 10% or 10% of unstimulated or stimulated with 0.4 μg / mL of SEB. The plate was incubated at 37°C for 5 days before analysis.

[0261] After centrifuging the plates, the supernatant was removed and the pellet was washed once with PBS 1x. Add 100 μL of diluted fixable viability dye solution Live Dead Aqua (ThermoFischer) was added, and the cells were incubated at 4°C. Incubated for 15 minutes and then washed with PBS, 0.5% BSA. Anti-CD4 AP C-Vio770 (Miltenyi Biotech;1 / 50), anti-CD3 Pac ific Blue (Becton Dickinson; 1 / 100), anti-CD8α Premixed antibody containing APC (Becton Dickinson; 1 / 100) The cocktail was added to the pellet. The cells were incubated on ice for 20 minutes before being resuspended in PB. Wash with S and then use MacsQuant10 (Miltenyi Biotech) Flow cytometry analysis was performed.

[0262] 7A-7E show the results of 2C8 at high (10 μg / mL) or low (1 μg / mL) saturation concentrations. v130, a high concentration (10 μg / mL) of a non-CD8 binding VHH (Lys2), or P Proliferation assay using CFSE-labeled human PBMCs (n=3) in the presence of BS (vehicle). The results are shown in Table 7. Without stimulation (culture medium, Figure 7A), 2C8.v130 also inhibited Lys 2-VHH also did not induce background proliferation in the three donors tested. Addition of VHHs inhibits CD8 + Non-specific binding, whether or not binding to cells Anti-CD3 / CD28 polyclonal stimulation shows no induction of T cell activation. (Figure 7B) showed optimal CD8 + T cell proliferation was obtained (dilution CFS Neither the addition of 2C8.v130 nor the addition of Lys2 VHH increased the T cell proliferation rate (more than 90% of the T cell proliferation rate). The superantigen SEB did not affect cell activation. Although SEB induces cross-linking of the nuclei, the addition of SEB to the samples results in high cell death and therefore suboptimal proliferation rates. However, in the case of a single donor, No significant difference was observed between the different conditions (Fig. 7C). To mimic TCR / CD8 complex association, a CEF peptide pool was used to stimulate TCR specific CD8 + However, the proliferation rate was similar to that in culture medium conditions. (Fig. 7D) and none of the donors tested had detectable reactive CD8 + T cells This confirmed the absence of VHH-induced background proliferation. Similarly, in the case of TLR4 stimulation of monocytes (LPS condition), the addition of VHH did not result in indirect stimulation. No significant T cell proliferation was observed (Fig. 7E).

[0263] Figures 8A-8D show growth assays using 10% FBS or autologous donor plasma as culture medium. The results of the assays were compared. Autologous donor plasma samples were obtained and maintained under physiological conditions before PBMC isolation. These were used in proliferation assays to mimic the conditions observed in the unstimulated donors. No differences were observed between experiments using % FBS medium or autologous donor plasma (Figure 8A and 8B). Thus, even in the presence of soluble human plasma factors, VHHs were able to express IgG over background It does not induce proliferation of HIV-1-associated T cells.

[0264] [Example 2] Evaluation of 2C8v144 VHH for molecular imaging Labeling of 2C8 VHH 18 F control VHH and 18 RESCA (Restrictive Complexing Agent) Modification containing anti-CD8 VHH Decorative VHH and 18 The production procedure for F-AlF-RESCA modified VHHs was previously described. The protocol was based on the protocol (Cleeren F. et al. Nature Protocols 13, 2330-2347 (2018)). An exemplary RESCA has the chemical structure of formula (I): Gate VHHs with sodium acetate or methionine and N-acetyl-tryptophan. in a reaction medium consisting of either sodium acetate with 18 F-Fluoride mixture The reaction mixture was diluted with histidine, methionine, and N- Acetyltryptophan and sucrose-conditioned formulation buffer. As a control, the formulation buffer was replaced with phosphate buffered saline. Conditioned with water. 18 To limit the dissociation rate of F-AlF To achieve this, the pH and temperature of the histidine formulation buffer were carefully controlled. Protein concentration, protein purity, and radiochemical purity were determined by SE-HPLC, and target Assay binding (and, if necessary, immunoreactive fractions) by SE-HPLC and SPR did.

[0265] 2C8v145 VHH used 18 F control VHH and 2C8v144 were used 18F anti-CD8 VHH were obtained with radiochemical yields of 40-60% (without decay correction). The final product exhibited a specific activity range of 3.0-8.0 Ci / μmol with a radiochemical purity of 95%. It exceeds 18 In the case of F anti-CD8 VHH, the immunoreactive fraction was greater than 94%.

[0266] Without being bound by theory or hypothesis, it is believed that anti-CD8 VHHs can be conjugated to radionuclide labels. Oxidation of one or more VHH residues, such as tryptophan, results in the oxidation of CD 8) May lead to reduced binding capacity. Conjugation reaction buffer, purification buffer , and / or in the formulation buffer, methionine and / or N-acetyltryptophan The use of antioxidant compounds such as α- and β-actin can reduce oxidation of VHH residues and thereby enhance their functionality. This can improve the yield of specifically labeled anti-CD8 VHH.

[0267] Chimeric CD8 in mice + PET imaging of tumor xenografts 18 Sensitivity and dynamic range of F-anti-CD8 VHH chimeras in mice PET imaging of CD8+ tumor xenografts was performed to assess this.

[0268] Briefly, a CD8-expressing human T-cell leukemia cell line (DSMZ, Germany) HPBALL was characterized using Daud, a CD8-deficient human lymphoma cell line (DSMZ, Germany). Mixed with i in various ratios, 18 F - for PET imaging using anti-CD8 VHH Chimeric tumors with various CD8 concentrations were generated. Briefly, female CB17.SCID.bg 10 million of each in a 50:50 mix of HBSS:Matrigel was injected into the dorsal thoracic region of the mouse. The cells were subcutaneously inoculated. The tumor size was approximately 400 mm 3 When it reaches 18 F-anti-CD 8 VHHs were injected into animals via the tail vein and analyzed by Inveon PET / computed tomography ( CT scanner (Siemens Preclinical Solutions, I The subjects were subjected to dynamic 60-minute PET scans at 100 Hz (nc.).

[0269] After PET imaging was completed, chimeric tumors were excised from the mice to assess CD8 expression. GentleMACS Octo Dissociation according to the manufacturer's protocol The resulting cells were then dissociated using a PBS or (Miltenyi Biotec). The suspension was passed through a 70 μm cell strainer (Corning) to remove aggregates. Tumor cells were then counted and 300,000 cells were cultured in the presence of 100 μL RPMI medium. The cells were plated in a round-bottom 96-well plate in the presence of 20 nM Alexa Fluor 470. FLUOR® 647-tagged OKT8 anti-CD8 antibody (60 min, 4°C) and Sy Tox Orange dead cell stain (15 min, 4°C) (Thermo Fisher Scientific The cells were washed and then stained with FACS Calibur flow cytometer. CD8 in tumors analyzed by cytometer + The percentage of cells was determined.

[0270] As shown in Figure 9, 18 F-anti-CD8 VHH inhibited CD8 + PET imaging also revealed that HPBALL cells express CD8 + Allows clear visualization of tumor cells The results also showed that PET uptake (ID% / g) and CD8 + Concentration of HPBALL cells Based on previous FACS data, HPBAL Each L cell contains approximately 55,000 to 85,000 copies of CD8 molecules, and naive CD8 8 + Each T cell is estimated to have 200,000 to 300,000 copies of the CD8 molecule. did. 18 F-anti-CD8 VHH induces low levels of C (sensitivity of approximately 1–30 nM) in tumor cells. It is a highly sensitive imaging agent that can detect D8 expression.

[0271] PET imaging of TALL1 tumor xenografts in mice 18 F-anti-CD8 VHH and 89 Zr-single arm ("OA") - anti-CD8 antibody ( (See International Patent Application Publication No. WO2019 / 033043) TALL1 tumor xenografts were imaged in mice. TALL1 is a tumor-associated protein that encodes low-CD8-expressing leukemia. Based on previous FACS data, TALL1 cells each produce approximately 12,000 It was estimated to have ~15,000 copies of the CD8 molecule.

[0272] Briefly, the right flank of female CB17.SCID.bg mice was inoculated with HBSS:Matrigel. Ten million TALL1 cells were inoculated subcutaneously in a 50:50 mix of TALL1 cells. Approximately 400m 3 Once this was achieved, animals were grouped for PET imaging. 89 Zr- For imaging with OA-anti-CD8 antibodies, animals were injected via the tail vein and then imaged at 0, 1, 2, and 3 sites. Static PET scans were performed on days 1 and 5. 18 Imaging with F-anti-CD8 VHH showed Animals were injected via the tail vein and dynamic 60-minute PET scans were performed as before.

[0273] Region of interest (ROI) measurements were performed using IRW software (Siemens Preclini Microscopy was performed on multiple axial slices of tissue using a microscope (microscope) based on the MRI software (microscope). The decay-corrected signal intensity of the organ was calculated as the equivalent of 1 cc in 1 gram of soft tissue. It was assumed and measured as a percentage of injected dose per gram (ID% / g).

[0274] 18 F-anti-CD8 VHH rapidly transformed low-CD8-expressing TALL1 xenograft tumors within 1 hour. As shown in Figure 10, CD8-expressing TALL1 Xenograft tumors could be clearly visualized 90 minutes after injection, with tumor densities as high as 14. The blood ratio was achieved. 18 Imaging with F-anti-CD8 VHHs takes place over 0.5–4 hours. In comparison, 89 Zr-OA-anti-CD8 antibody has a longer-lasting effect This is suitable for meaningful imaging at this time point, i.e., 1 to 5 days after injection. 18 F-anti-CD8 V Due to its small size, HH penetrates tissues very quickly and exhibits rapid renal clearance. and perform an additional PET scan (e.g., FDG PET) on the same patient later that day. and facilitate a repeat CD8 scan the next day, or as early as the next day. 18 F labeling ( or 68 It is compatible with other radiolabeled compounds such as Ga, which reduces the radiation burden posed to the patient. This allows imaging procedures using relatively low levels of anti-CD8 VHH, thus allowing for the detection of a subset of human patients. Additional scans may be performed throughout the treatment course within general dosimetry guidelines. For example, 18With F-anti-CD8 VHH, typically several months or re-imaging the same patient up to approximately five times over the course of several years of treatment and follow-up. It will be possible to

[0275] PET imaging studies in rhesus monkeys In rhesus monkeys 18 Imaging experiments with F-anti-CD8 VHH were performed, typically with C We determined whether uptake in tissues rich in D8 could be detected. 5 kg) containing 64 micrograms of radiation containing 1.2 mCi 18 F-anti-CD8 CD8-rich tissues, such as lymph nodes, thymus, and spleen, were isolated from the injected VHHs. For example, the upper image in Figure 11 shows the image obtained within 1 hour of injection. PET MIP images after 10 min are shown. 18 F PET 1 hour after injection of control VHH In the MIP image, no CD8-rich tissue was visible (Fig. 11, bottom). Only clearance was significant.

[0276] [Example 3] Immunotherapy for cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease Methods for using CD8 imaging to determine the efficacy of 18 Using a CD8 binding agent as described herein, such as an F-anti-CD8 VHH, CD8 + Such imaging can be used to assess tumor and lymph node infiltration by cells. Identify immune phenotypes that predict patient prognosis and / or response to immunotherapy. Such imaging can be used to detect, for example, diseased tissue (e.g., tumors) and other lymph nodes. CD8 +Determine the ubiquity of T cells. Such imaging can be used to investigate the role of T cells in cancer, autoimmune diseases, and diseases or conditions (such as arthritis, colitis, or celiac disease), transplant rejection, or graft Immunotherapeutic agent or agents comprising one or more immunotherapeutic agents for patients with host-versus-host disease Select a combination therapy.

[0277] In all embodiments disclosed herein, immunotherapy for cancer patients includes, for example, Monoclonal antibodies, T-cell and tumor-associated protein-binding duplexes for treating specific antibodies, bispecific antibodies that bind to NK cells and tumor-associated proteins, cytokines Cancer immunotherapy, CAR-T cell therapy, non-specific cancer immunotherapy and adjuvants, and immune chain therapy any anti-PD1 or anti-PDL1 agent disclosed herein, such as a checkpoint inhibitor. Examples of bispecific antibodies that bind to T cells and tumor-associated proteins include anti-CD40 antibodies. 3 Bispecific antibodies that bind to NK cells and tumor-associated proteins Examples of the specific antibodies include anti-CD16 (FcgammaRIII) bispecific antibodies, anti-C D16A bispecific antibody, anti-CD56 bispecific antibody, anti-NKp46 bispecific antibody, and and any other NK cell-binding bispecific antibody.

[0278] In some embodiments, the CD8 binding agent (e.g., 18 F-anti-CD8 VHH) As described herein, cancer, an autoimmune disease or condition, transplant rejection, or graft versus host Treatment, diagnosis, prognosis, companion diagnostics, and progression / remission monitoring of primary and secondary diseases It can be used for

[0279] In some embodiments, the CD8 binding agent (e.g.,18 F-anti-CD8 VHH) Immunotherapeutic agents for diseases (cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease) It can be used to image subjects who have experienced treatment failure for their primary disease, and the imaging results can help determine whether they are cured. For example, a subject may receive an atezolizumab combination and respond to treatment. Imaging results may not be available if the subject is CD8 + tumor cells lost or still as CD8 + tumor cells, but the therapy is exhausted or the tumor is CD8+ It can be determined whether the compound is no longer effective against the cells.

[0280] [Example 4] for using CD8 imaging for microbiome research and immunophenotyping method 18 Using a CD8 binding agent as described herein, such as an F-anti-CD8 VHH, CD8 + Tumor and lymph node infiltration by cells can be assessed. using microbiota to predict patient prognosis and / or response to cancer immunotherapy Identify the immune phenotypes underlying the iomic signatures.

[0281] moreover, 18 Using the CD8 binding agents described herein, such as F-anti-CD8 VHHs, Using CD8 + Mycobacterium leukaemia (MYC) is associated with specific systemic patterns of T cell biodistribution. Such imaging can be used to identify black biome signatures, e.g. For example, CD8 in tumors and other lymph nodes + This determines the ubiquity of T cells. Imaging allows for the detection of highly noisy or weakly correlated results. The most robust microbiome biomarkers will be selected.

[0282] Resident gut bacteria may influence patient response to cancer immunotherapy. See Gopalakrishnan et al. (2018) Science. 359(6371): 97-103. This will allow us to identify key microbial strains associated with patient response to cancer immunotherapy. and may be useful in identifying appropriate treatment regimens for cancer patients. 18 F-anti-CD8 VH The CD8 binding agents described herein, such as H, can be used to treat immunotherapy (e.g., Microbiome profiles correlate with patient response to immunotherapy (discussed in this paper). It is possible to identify the characteristics of the gut microbiota (e.g., gut microbiota composition).

[0283] Briefly, gut microbiome samples (e.g., fecal samples) can be used to treat immune-mediated diseases (e.g., The immunotherapy may be obtained from a cancer patient who will be receiving immunotherapy (e.g., immunotherapy as described elsewhere herein). 18 F- The CD8 binding agents described herein, such as anti-CD8 VHHs, can be administered to patients undergoing cancer immunotherapy. Each patient was administered CD8 + Cells were used to assess each patient's tumor and lymph node involvement. Each patient then receives a cancer immunotherapy (e.g., an immunotherapy described herein). . 18 The CD8 binding agents described herein, such as F-anti-CD8 VHHs, can be administered to cancer immunotherapy patients. The patient was given another dose after treatment, and once again, CD8 + Tumor and lymph node counts for each patient Assess infiltration. Evaluate the level of CD8 infiltration in tumors and lymph nodes of patients after immunotherapy. and each patient's microbiome profile (e.g., gut microbiome sample) Determine the types of microorganisms present in the tumor and the abundance of each type of microorganism. CD8 to lymph nodes + Significant changes in gut microbiome samples from patients with T cell infiltration Identify the microbial strains.

[0284] CD8 to lymph nodes and / or tumor + Important microbial strains in patients with T cell infiltration Once identified, microbiome medicines containing important microbial strains can be developed using microbiome-based drugs obtained from such patients. The microbiome medicine is made from donor feces and delivered to lymph nodes or tumors by CD8 + Administered to patients who do not demonstrate T cell infiltration. Alternatively, administer to patients who demonstrate T cell infiltration in lymph nodes and / or tumors. CD8 + Donor feces collected from patients showing T cell infiltration were used to identify lymph nodes and / or or CD8 to tumors + For patients without T cell infiltration, fecal microbiota transplantation (FMT) In some embodiments, the FMT or microbiome medicine is administered to a cancer immunotherapy patient. CD8 to lymph nodes and / or tumor at the time of therapy + Patients without infiltration were treated with cancer immunotherapy. converting patients into those who respond to the treatment.

[0285] In some embodiments, CD8 imaging is performed prior to FMT or after microbiome drug administration. Prior to CD8 + Performed in patients without infiltration Patients will receive immunotherapy after FMT or microbiome medicine. , FMT or microbiome drugs may increase CD8 + infiltration Imaging is performed on patients to determine whether the tumor causes an increase in blood pressure. In embodiments, C in response to cancer immunotherapy treatment after FMT or other microbiome drugs. D8 + If increased infiltration is observed, FMT or other microbiome drugs have been successful It is considered to be.

[0286] The CD8 imaging agent used in conjunction with microbiome research and discovery is wt2C8 VHH, 2C8v130 VHH, 2C8v142 VHH, or 2C8v144 VHH was used 18 F - any CD8 binding VHH described herein, such as an anti-CD8 VHH The compound may be a sexually active agent.

[0287] In some embodiments, the cancer immunotherapy is an immune checkpoint inhibitor. In embodiments, the cancer immunotherapy is a T cell targeted therapy. Therapies include T cell bispecific, trispecific, or multispecific antibodies or their antigen-binding fragments. In some embodiments, the cancer immunotherapy is an NK cell targeted therapy. In embodiments, the NK cell targeted therapy is a bispecific, trispecific, or multispecific antibody. or an antigen-binding fragment thereof.

[0288] In some embodiments, 18 F - CD8 binding as described herein, such as anti-CD8 VHH CD8 imaging using active agents, such as checkpoint inhibitors, or CD16 or tumor and and lymph node CD8 + Such imaging can be used to assess invasion. and the like, such as checkpoint inhibitors or CD16 or CD3 targeting moieties. Determine microbiome biomarkers associated with the efficacy of immune-modulating molecules.

[0289] The checkpoint inhibitor used in this example was any checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is anti-PD1 or anti-PDL1 In some embodiments, the checkpoint inhibitor is atezolizumab (TEC ENTRIQ (registered trademark).

[0290] The immunomodulatory molecule may be any molecule that affects CD8 cell proliferation and infiltration. Examples include antibodies that bind to CD3 and tumor-associated antigens, and antibodies that bind to CD16 and tumor-associated antigens. and T cell bispecific molecules such as molecules that bind to related antigens.

[0291] Illustrative embodiments and examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention. However, it is not intended to limit the scope of this application. Indeed, those skilled in the art will recognize that the present invention is not limited to the above-described embodiments. Various modifications in addition to those shown and described in will be apparent from the foregoing description. It is intended to fall within the scope of the appended claims.

Claims

1. A CD8-binding agent comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain), , K of about 1 nM or lower Ｄ CD8 binding activity that specifically binds to human CD8 Agent.

2. k of about 0.002 / sec or lower ｏｆｆ The method of claim 1, which binds to human CD8 at The CD8 binding agent described above.

3. K of about 1 nM or lower Ｄ 1 or 2, which binds to cynomolgus monkey CD8 at 3. The CD8 binding agent according to claim 2.

4. k of about 0.004 / sec or lower ｏｆｆ binds to cynomolgus monkey CD8 at Item 4. The CD8 binding agent according to any one of Items 1 to 3.

5. CD8 ＋ 5. The method of claim 1, wherein the antibody neither stimulates nor inhibits T cell activation. A CD8 binding agent of

6. CD8 ＋ The CD8 binding antibody of any one of claims 1 to 5, which does not induce T cell proliferation. Sexually active agent.

7. CD4 ＋ 7. The CD8-binding molecule of any one of embodiments 1 to 6, which does not bind to T cells. Agent.

8. The CD of any one of claims 1 to 7, wherein the VHH domain is a llama VHH. 8-binding agents.

9. The CD of any one of claims 1 to 8, wherein the VHH domain is humanized. 8-binding agents.

10. The VHH domain comprises Arg25, Lys42, Gln44, Val45, Leu4 6, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Ar Human CD8α gene containing g93, Leu94, Gly95, Asp96, and Thr97 10. A method according to claim 1, wherein the amino acid numbering is according to SEQ ID NO:

13. The CD8 binding agent according to any one of claims 1 to 4.

11. The VHH domain has a complementarity determining region (CDR) comprising the amino acid sequence of SEQ ID NO: 6 or 7 ( CDR1 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and CDR2 comprising the amino acid sequence of SEQ ID NO: 10 to 12.

11. The CD8 antibody of claim 10, comprising a CDR3 comprising any one of the amino acid sequences of 12. Synergistic agent.

12. The VHH domain is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 8 a DR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9 a DR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 9 a CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 12. The CD8 binding agent of claim 11, comprising:

13. the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; and CDR2 comprising the amino acid sequence of SEQ ID NO:

12.

12. The CD8 binding agent of claim 11.

14. 1 to 3, wherein the VHH domain comprises L49A and the numbering is according to Kabat numbering.

14. The CD8 binding agent of any one of 13.

15. The VHH domain comprises a V89T substitution, a T110Q substitution, a S112Q substitution, and an A1 14 additions, and the numbering is 15. A CD8 binding agent according to any one of claims 1 to 14, according to the at numbering.

16. The VHH domain comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4.

16. The CD8 binding agent of any one of 1 to 15.

17. 17. The CD8 binding agent of any one of claims 1 to 16, which does not contain an Fc region.

18. An isolated antibody encoding the CD8 binding agent of any one of claims 1 to 17. Nucleic acid.

19. 19. An expression vector comprising the nucleic acid of claim 18.

20. 20. A host cell comprising the nucleic acid of claim 18 or the expression vector of claim 19.

21. eukaryotic cells, such as mammalian cells (e.g., Expi293 cells), or prokaryotic cells.

21. The host cell of claim 20.

22. 1. A method for making a CD8 binding agent, comprising: a) culturing the host cell of claim 20 or 21 under conditions in which the agent is produced; and b) recovering the CD8 binding agent produced by the host cell A method comprising:

23. 18. Any of claims 1 to 17, wherein the VHH domain is conjugated to a label. The CD8 binding agent of any one of claims 1 to 4.

24. 24. The CD8 binding antibody of claim 23, wherein the label is a fluorescent dye, a radionuclide, or an enzyme. Synergistic agent.

25. 25. The CD8 binding agent of claim 24, wherein the label is a radionuclide.

26. The radionuclide is １８ F. ８９ Zr, ９９ｍ Tc, ６７ Ga, ６８ Ga, ６４ Cu, ５２ Mn, １１１ In, or １２４ 26. The CD8 binding agent of claim 25, wherein 。

27. wherein the VHH domain is conjugated to a label via a chelating moiety.

27. The CD8 binding agent of any one of paragraphs 23 to 26.

28. the chelating moiety is covalently linked to the VHH domain via a lysine residue; 28. The CD8 binding agent of claim 27,

29. The label forms a complex with a metal, and the complex is chelated by the chelating moiety.

29. The CD8 binding agent of claim 27 or 28.

30. The sign １８ 30. The method of claim 29, wherein the metal is aluminum. Binding agents.

31. The chelating moiety has the formula (I): 【Chemistry 1】 31. The CD8 binding agent of claim 30, which is a compound of the formula:

32. Subject's CD8 ＋ 1. A method for detecting a cell, comprising: a) administering to the subject a labeled CD8 binding agent of any one of claims 23 to 31 administering; and b) combining the labeled CD8 binding agent with the subject's CD8 ＋ Steps to detect binding to cells P and detecting said binding comprises detecting CD8 ＋ indicating the presence of cells, method.

33. the labeled CD8 binding agent and the subject's CD8 ＋ detecting binding to cells , the subject's CD8 ＋ 33. The method of claim 32, comprising imaging the cells.

34. the subject's CD8 ＋ Imaging the cells includes performing positron emission tomography (PET) on the subject. scan or positron emission tomography / computed tomography (PET / CT) scan 34. The method of claim 33, comprising performing

35. The CD8 ＋ The cells are CD8 ＋ T cells or CD8 ＋ tumor cells from claim 32 34. The method of any one of claims 34 to 34.

36. The detecting step is performed within about 1 day or less after the administering.

36. The method of any one of claims 32 to 35,

37. 37. The method of any one of claims 32 to 36, repeated one or more times.

38. 38. The method of claim 37, wherein the administration is repeated about 1 day after the previous administration of the CD8 binding agent. How to do it.

39. 39. The method of claim 37 or 38, which is repeated 1 to 4 times per year.

40. 40. The method of any one of claims 37 to 39, repeated for a period of more than one year. How to do it.

41. The method according to any one of claims 32 to 40, having a sensitivity of about 1 nM to about 30 nM. Law.

42. 42. The method of any one of claims 32 to 41, wherein the subject is a human or a non-human primate. How to post.

43. 43. The method of claim 42, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey. 。

44. 44. The method of any one of claims 32 to 43, wherein the subject has cancer.

45. the subject has an autoimmune disease or condition, transplant rejection, or graft-versus-host disease; 44. The method of any one of claims 32 to 43.

46. Predicting the response of subjects with cancer to immunotherapy, cell therapy, or cancer vaccines A method for measuring a) administering to the subject a labeled CD8 binding agent of any one of claims 23 to 31 administering; and b) measuring the CD8 binding agent and the CD8 binding activity of the tumor tissue of the subject; ＋ Binding to T cells Detecting Steps detecting said binding, wherein said subject is a patient receiving said immunotherapeutic agent, said cell therapy, or said cancer indicating a high likelihood of responding to the vaccine, method.

47. (c) administering a therapeutically effective amount of the immunotherapeutic agent, the cell therapy agent, or the like to the subject in whom the binding is detected. or administering the cancer vaccine.

47. The method of claim 46, further comprising:

48. 1. A method for monitoring disease progression in a subject having cancer, comprising: a) administering to the subject a labeled CD8 binding agent of any one of claims 23 to 31 administering; and b) measuring the CD8-binding agent and the tumor of the subject at a first time point and a second time point; Tissue CD8 ＋ Detecting binding to T cells A method comprising:

49. (c) administering to the subject a therapeutically effective amount of an immunotherapeutic agent, a cell therapy, or a cancer vaccine. and further comprising the step of: detecting CD8 in the tumor tissue at the second time point; ＋ T cell levels wherein the CD8 of the tumor tissue at the first time point ＋ higher than the level of T cells, 49. The method of claim 48.

50. have received or are receiving immunotherapy, cell therapy, or cancer vaccines, or have cancer 1. A method for monitoring the treatment progress of a subject, comprising: i) a compound according to claim 23 in combination with said immunotherapeutic agent, said cell therapy, or said cancer vaccine; 32. Administering to the subject a labeled CD8 binding agent of any one of claims 1 to 31. and ii) measuring the C of the labeled CD8 binding agent with the tumor tissue at a first time point and a second time point; D8 ＋ Detecting binding to T cells A method comprising:

51. The labeled CD8 binding agent is a compound that binds to the immunotherapeutic agent, the cell therapy, or the cancer vaccine. and the first time point is administered after administration of the labeled CD8 binding agent but before administration of the the second time point is prior to administration of the immunotherapeutic agent, the cell therapy, or the cancer vaccine; 50. after administration of the immunotherapeutic agent, the cell therapy, or the cancer vaccine. The method described below.

52. The immunotherapeutic agent, the cell therapy, or the cancer vaccine binds to the labeled CD8 binding antigen. and the first time point is administered before the administration of the immunotherapeutic agent, the cell therapy, or the after administration of a cancer vaccine and after administration of the labeled CD8 binding agent, 51. The method of claim 50, wherein the time point is after the first time point.

53. 53. The method of claim 46-47 and 49-52, wherein the immunotherapeutic agent is administered to the subject.

10. The method according to any one of claims 1 to 9.

54. The immunotherapeutic agent is an anti-PDL1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, a TIGIT antibody, antagonist, anti-CSF-1R antibody, anti-CSF-1R antagonist, anti-CEA antibody, anti CEA antagonist, anti-CTLA4 antibody, CTLA4 antagonist, anti-OX40 antibody , or an OX40 agonist.

55. 55. The method of claim 54, wherein the immunotherapeutic agent is an anti-PD-L1 antibody.

56. 56. The method of claim 55, wherein the anti-PD-L1 antibody is atezolizumab.

57. The anti-PD-L1 antibody is administered in combination with one or more therapeutic agents.

57. The method according to claim 55 or 56.

58. The one or more therapeutic agents are TARCEVA® (erlotinib), ZERONA® (E2), LBORAF® (vemurafenib), GAZYVA® (obinutuz) (bevacizumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTEL LIC® (cobimetinib), ALECENSA® (alectinib) , KADCYLA® (ado-trastuzumab emtansine), HERCEPTI N® (trastuzumab), PERJETA® (pertuzumab), Latuzumab, IFN-alpha, anti-CD40 agent, anti-OX40 antibody, OX40 agonist, The antibody is an anti-CSF-1R antibody, an anti-CEA antibody, an IDO inhibitor, or an anti-TIGIT antibody.

58. The method of claim 57.

59. The immunotherapeutic agent is selected from the group consisting of IL2, genetically modified IL2, IL15, and genetically modified IL15.

54. The method of claim 53, wherein the cytokine is a cytokine such as

60. The immunotherapeutic agent may comprise a dendritic cell module, such as a dendritic cell activator or a dendritic cell growth factor.

54. The method of claim 53, wherein the activator is a methyltransferase.

61. The immunotherapeutic agent is a dual molecule that specifically binds to CD3 or CD16, such as CD16A.

54. The method of claim 53, wherein the antibody is a specific antigen-binding molecule.

62. 52. The method of claim 46, wherein the cancer vaccine is administered to the subject.

10. The method according to any one of the preceding claims.

63. 63. The method of claim 62, wherein the cancer vaccine is a personalized cancer vaccine (PCV). 。

64. 53. Any of claims 46-47 and 49-52, wherein the cell therapy is administered to the subject. The method according to any one of claims 1 to 4.

65. 65. The cell therapy of claim 64, wherein the cell therapy is CAR-T or neoantigen-specific T cells. method.

66. Immunotherapy of subjects with autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease 1. A method for predicting responsiveness to a therapeutic agent, comprising: a) administering to the subject a labeled CD8 binding agent of any one of claims 23 to 31 administering; and b) measuring the CD8 binding agent and CD8 in the diseased tissue of the subject; ＋ Binding to T cells Detecting Steps wherein detecting said binding indicates that said subject is likely to respond to said immunotherapeutic agent. vinegar, method.

67. (c) administering a therapeutically effective amount of the immunotherapeutic agent to the subject in whom the binding is detected. Top 67. The method of claim 66, further comprising:

68. Disease progression in subjects with autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease 1. A method for monitoring a a) administering to the subject a labeled CD8 binding agent of any one of claims 23 to 31 administering; and b) combining the labeled CD8 binding agent with the subject's disease at a first time point and a second time point. CD8 in diseased tissue ＋ Detecting binding to T cells and detecting CD8 from the first time point and the second time point. ＋ Increased T cells contribute to autoimmunity indicating progression of disease, transplant rejection, or graft-versus-host disease; method.

69. (c) administering to said subject a therapeutically effective amount of an immunotherapeutic agent. and further comprising measuring CD8 of the diseased tissue at the second time point. ＋ The level of T cells is CD8 of the diseased tissue at a first time point ＋ Lower than T cell levels 69. The method of claim 68.

70. have received or are receiving immunotherapy, have an autoimmune disease or condition, transplant rejection, or A method for monitoring the treatment progress of a subject with graft-versus-host disease, comprising: i) a labeled CD8 antibody according to any one of claims 23 to 31 in combination with said immunotherapeutic agent; administering a binding agent to the subject; and ii) measuring the CD8 binding agent and the CD8 binding agent in the diseased tissue at a first time point and a second time point; D8 ＋ Detecting binding to T cells A method comprising:

71. the labeled CD8 binding agent is administered before the immunotherapeutic agent, and the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, 71. The method of claim 70, wherein the point is after administration of the immunotherapeutic agent.

72. the immunotherapeutic agent is administered before the labeled CD8 binding agent, and the first time point is after administration of the immunotherapeutic agent and after administration of the labeled CD8 binding agent, 71. The method of claim 70, wherein the second time point is after the first time point.

73. the labeled CD8 binding agent and the subject's CD8 ＋ The step of detecting binding to cells , the subject's CD8 ＋ 73. Any one of claims 46 to 72, comprising imaging T cells. The method described in the first paragraph.

74. the subject's CD8 ＋ Imaging the cells includes performing positron emission tomography (PET) on the subject. scan or positron emission tomography / computed tomography (PET / CT) scan 74. The method of claim 73, comprising performing

75. Claims 48-65 and 68-7, wherein the subject is monitored for at least one year.

4. The method according to any one of claims 1 to 3.

76. To identify gut microbial strains associated with response to immunotherapy treatment. The method of a) obtaining gut microbiome samples from a population of subjects with cancer, The population includes subjects who are responsive to treatment with the immunotherapeutic agent and subjects who are resistant to treatment with the immunotherapeutic agent. including subjects who are not responsive to treatment with the b) the gut microbiome sample of the subject responsive to the treatment and the treatment analyzing the gut microbiome sample from the subject who is not responsive to the treatment; and c) identifying gut microbial strains associated with said subject that are responsive to said treatment; Top and the responsiveness is determined by the labeled CD8 binding agent of any one of claims 23 to 31. and CD8 in the tumor tissue of the subject. ＋ by detecting binding to T cells, detecting binding indicates that the subject is responsive to the immunotherapeutic agent. method.

77. A microbiome, including gut microbial strains associated with responsiveness to said immunotherapeutic agent.

77. The method of claim 76, further comprising preparing a medicament based on the compound.

78. The immunotherapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody such as atezolizumab.

78. The method of claim 76 or 77.

79. 32. A kit comprising the labeled CD8 binding agent of any one of claims 23 to 31.

80. A method for preparing a labeled CD8 binding agent, comprising:

18. A CD8 binding agent according to any one of claims 17 to 18, wherein the VHH domain is conjugated to the VHH domain of the CD8 binding agent according to any one of claims 17 to 18. providing a conjugate; and reacting said conjugate with １８ Contains F and contacting the labeled CD8 binding agent with an aluminum fluoride complex containing the compound. wherein the chelating moiety comprises a group having the formula (I): 【Chemistry 2】 The method is a compound of

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